Inhaled nitric oxide increases surfactant protein gene expression in the intact lamb
Regan B. Stuart,1
Boaz Ovadia,1
Vincent V. Suzara,1
Patrick A. Ross,1
Stephan Thelitz,2
Jeffrey R. Fineman,1,3 and
Jorge A. Gutierrez1
Departments of 1Pediatrics and
2Surgery, and 3The
Cardiovascular Research Institute, University of California, San Francisco,
California 94143
Submitted 6 August 2002
; accepted in final form 15 May 2003
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ABSTRACT
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Inhaled nitric oxide (iNO) is used to treat a number of disease processes.
Although in vitro data suggest that nitric oxide (NO) alters surfactant
protein gene expression, the effects in vivo have not been studied. The
objective of this study was to evaluate the effects of iNO on surfactant
protein (SP)-A, -B, and -C gene expression in the intact lamb. Thirteen
4-wk-old lambs were mechanically ventilated with 21% oxygen and received iNO
at 40 ppm (n = 7) or vehicle gas (n = 6) for 24 h.
Peripheral lung biopsies were obtained at 0, 12, and 24 h and analyzed for
surfactant mRNA, protein, and total DNA content. Inhaled NO increased SP-A and
SP-B mRNA content by 80% from 0 to 12 h and by 78 and 71%, respectively, from
0 to 24 h. There was an increase in SP-A and SP-B protein content by 45% from
0 to 12 h, and a decrease by 70 and 65%, respectively, from 0 to 24 h. DNA
content was unchanged. The mechanisms and physiological effects of these
findings warrant further investigation.
surfactant proteins
NITRIC OXIDE (NO) is a free radical that serves as an important
cellular messenger affecting vascular tone
(14), platelet adhesion
(17), immunological responses
(8), and neurotransmission
(27). Endogenous NO is
synthesized in endothelial cells from arginine and activates soluble guanylyl
cyclase, which catalyzes the formation of cGMP, relaxes smooth muscle cells,
and activates protein kinases
(14). Exogenously administered
inhaled NO (iNO) has been shown clinically to be a potent, selective pulmonary
vasodilator (11,
28). It is proven effective in
reducing pulmonary vascular resistance in newborns with persistent pulmonary
hypertension (5,
6,
10,
22) and in children with
various congenital heart diseases who suffer from postoperative pulmonary
hypertension (2,
19). Recently, iNO has also
been used to improve oxygenation in acute lung injury by increasing
ventilation and perfusion matching, thereby decreasing the shunt fraction
(20,
23).
However, NO has been shown to damage surfactant proteins and alter their
function (13). A recent in
vitro study by Ayad and Wong
(4) demonstrated that NO
negatively affected pulmonary cell viability, surfactant protein expression,
and modulated surfactant protein (SP)-A gene expression in a human lung tumor
cell line (H441) known to have similar cellular properties as distal lung
epithelium. Matalon et al.
(18) ventilated newborn lambs
with multiple concentrations of NO through tracheostomy tubes in an open
ventilation circuit. Bronchoalveolar lavage (BAL) fluid from the lambs exposed
to high doses of iNO (80-200 ppm) for 6 h showed damage to SP-A apoproteins
and a decreased ability to aggregate lipids in vitro. Evaluation of BAL fluids
from both rats and piglets after prolonged NO exposure (24-48 h) at 100 ppm
demonstrated increased pulmonary inflammation, damage to epithelial fluid
lining, and alteration in the surface active properties of surfactant
(15,
21).
On the other hand, several studies have shown NO to have beneficial effects
on surfactant function as well. In a study evaluating BAL fluid from rabbits
treated with low-dose iNO (20 ppm), Sison et al.
(25) showed that NO exposure
improved alveolar surface activity, increased large surfactant aggregates, and
was beneficial to gas exchange. NO at higher doses (80 ppm) has been shown to
enhance the surface activity of surfactant
(16).
The aim of our study was to determine the effect of iNO at a moderate dose
(40 ppm) on SP-A, -B, and -C gene expression in the intact lamb. To this end,
we ventilated 4-wk lambs with 21% oxygen and 40 ppm of iNO for 24 h.
Intermittent lung biopsies were sampled for the determination of SP-A, -B, and
-C mRNA content and SP-A and -B protein content.
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METHODS
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Experimental protocol. Thirteen 4-wk-old lambs were anesthetized
with intravenous infusions of ketamine hydrochloride (1 mg ·
kg-1 · min-1) and diazepam
(0.002 mg · kg-1 · h-1, intubated with a
6.0-7.0-mm outer diameter endotracheal tube, and mechanically ventilated with
a Healthdyne pediatric time-cycled, pressure-limited ventilator (Healthdyne,
Marietta, GA). The lambs were ventilated with an average tidal volume of 10
cc/kg as measured intermittently by a bedside COSMO monitor
(Respironics/Novametrics, Wallingford, CT). Heart rate and systemic blood
pressure were monitored continuously to ensure adequate anesthesia. The lambs
were ventilated with 21% oxygen, and the rate was adjusted to maintain a
PaCO2 between 35 and 45 Torr. A midsternotomy incision
was performed, and the thoracic structures were exposed. Three single-lumen
polyurethane catheters were inserted into the left and right atrium (LA and
RA, respectively) and main pulmonary artery (PA). An ultrasonic flow probe
(Transonics Systems, Ithaca, NY) was placed around the left PA to measure left
pulmonary blood flow (Q). After a 60-min recovery, baseline hemodynamic
variables (LA, RA, PA, heart rate, systemic arterial pressure, and oxygen
saturations) were continuously monitored. A side-biting vascular clamp was
utilized to isolate peripheral lung tissue from a randomly selected lobe, and
the incision was cauterized. Approximately 300 mg of peripheral lung were
obtained. iNO (40 ppm) was then delivered in nitrogen into the inspiratory
limb of the ventilator (INOvent; Datex-Ohmeda, Andover, MA) in seven of the
lambs and was continued for 24 h. The other six lambs were ventilated, without
iNO, for 24 h. Peripheral lung biopsies were repeated at 12 and 24 h. Body
temperatures were monitored continuously and maintained between 38° and
40°C with a warming blanket. The midline incisions were approximated and
closure maintained with Kelly clamps.
At the end of the study period, the lambs were euthanized by an intravenous
injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union
City, CA). The committee on Animal Research of the University of California,
San Francisco (UCSF), approved all procedures and protocols.
Lung tissue preparation. Lung tissue biopsies were weighed for wet
weight values and snap frozen in liquid nitrogen. Tissue samples were stored
at -80°C until use.
Hemodynamic measurements. PA, LA, RA, and systemic pressures were
measured by Sorenson Neonatal Transducers (Abbot Critical Care Systems,
Chicago, IL). Mean pressures were obtained by electrical integration. Heart
rate was measured by a cardiotachometer triggered from the phasic systemic
arterial pressure pulse wave. Q was measured on an ultrasonic flow meter
(Transonics). All hemodynamic variables were recorded continuously on a Gould
multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic arterial
blood gases and pH were measured on a Radiometer ABL5 pH and blood gas
analyzer (Radiometer, Copenhagen, Denmark). Hemoglobin concentration and
oxygen saturation were measured by a hemoximeter (model 270, Ciba-Corning).
The pulmonary-to-systemic blood flow ratio was calculated using the Fick
principle.
Preparation of RNA, Northern blotting, and hybridization. Lung
tissue was pulverized and then briefly homogenized in RNA-STAT (Tel-Test,
Friendswood, TX). Total cellular RNA was extracted with phenol-chloroform,
precipitated with isopropanol, and quantitated spectrophotometrically. RNA
integrity was assessed by electrophoresis. Total RNA (10 µg/sample) was
separated electrophoretically on 1% agarose gels. Total RNA was transferred to
positively charged nylon membranes (BrightStar-Plus; Ambion, Austin, TX) and
cross-linked with ultraviolet light (UV Stratalinker 2400; Stratagene, La
Jolla, CA). Filters were probed with cDNAs for ovine SP-A, SP-B, and SP-C (a
kind gift of Dr. Phillip Ballard) and 18s rRNA, labeled with
[
-32P]dCTP (NEN Research Products, Boston, MA) by
random-primer second strand synthesis (Random Primer Labeling Kit; GIBCOBRL,
Gaithersburg, MD). Filters were prehybridized for 10 min in QuikHyb
hybridization solution (Stratagene) at 68°C and then hybridized in 10 ml
of QuikHyb solution containing 1.25 x 106 dpm/ml for 18 h.
Hybridized filters were washed under high stringency conditions and subjected
to autoradiography (Hyperfilm; Amersham CEA, Uppsala, Sweden). Radiolabeled
bands were quantified by volume integration of pixels measured by
phosphorimage analysis (Imagequant software; Molecular Dynamics, Sunnyvale,
CA). Filters were also probed for 18s rRNA as a control measure to ensure
equal loading of samples.
Protein measurement. Protein content was measured by the
bicinchoninic acid method (Pierce, Rockford, IL)
(26).
Lung tissue dry weight measurement. To estimate dry weight, we
dried a sample of peripheral lung tissue for 72 h in a vacuum oven at 86°C
and weighed it. Dry weight measurements were used in determining surfactant
protein and DNA content.
Quantification of SP-A and SP-B by dot-blot analysis. Lung tissue
was thawed at room temperature (RT) and homogenized to powder consistency and
dounced in 1 ml 50 mM NaHCO3 (pH 9). The samples were sonicated on
ice for 30 s each and centrifuged at 14,000 revolutions/min for 1 min. The
supernatant was removed, and protein content was measured as above. SP-A and
SP-B were assayed by quantitative dot blotting. Appropriate amounts of
supernatant were diluted 1:10 with 50 mM NaHCO3, pH 9. Duplicate
dots of serial dilutions were assayed on the same piece of nitrocellulose
(Bio-Dot Slot Format; Bio-Rad Laboratories, Richmond, CA). A gentle vacuum was
applied to the dot-blot apparatus until all the solution was pulled through.
The nitrocellulose was removed from the apparatus, and endogenous peroxidase
activity was quenched with 15% H2O2 for 5 min.
Nonspecific binding was blocked with a solution of 5% nonfat dry milk, 0.3%
bovine serum albumin, 0.4% gelatin, and 20 mM Tris-buffered saline (TBS), pH
7.4, at RT for 60 min. The blots were washed in Tris-buffered saline with 0.5%
Tween 20 (TBS-T), five washes for 5 min each, and probed separately for SP-A
and SP-B proteins. The blots were incubated in primary rabbit antibody
(1:3,000 dilution in TBS-T) against ovine sheep SP-A and SP-B (a kind gift of
Dr. Sam Hawgood, UCSF Medical Center, San Francisco, CA) for 30 min. The blots
were washed repeatedly in TBS-T over1hand then incubated in
peroxidase-labeled, secondary donkey antibody (1:2,000 dilution in TBS-T)
against rabbit Ig (Amersham CEA) for 20 min. Specificity and sensitivity of
these antibodies against sheep have previously been published
(12). The blots were washed
repeatedly in TBS-T over 1 h. Bound secondary antibody was detected by
exposure to Super Signal Dura (Pierce) for 5 min and enhanced
chemiluminescence. Relative light units were read in a plate luminometer
(Packard Instrument, Downer's Grove, IL).
DNA analysis. The DNA determination method described by Setaro and
Morley (24) was used. In
brief, DNA standards (50 µg calf thymus DNA/ml 1 N NH4OH, Sigma
D1501) and samples were dried at 60°C with N2 for
5-10
min. One hundred microliters of 3,5-diaminobenzoic acid (Sigma D1891, 0.3 g/ml
double distilled H2O) were added to each tube, vortexed, incubated
at 60°C for 5 min, vortexed, and incubated for 40 min. Samples were cooled
to RT for 5 min, and 1.4 ml 1 N HCl was added to each sample, vortexed, and
read by fluorimeter with excitation at 410 nm, emission at 520 nm, and slit
width of 0.5 mm (Fluorolog; ISA Instruments, Research Triangle Park, NC).
Values were normalized to the gram of dry lung weight.
Statistical analysis. Analysis of within-group and between-group
measurements were made by using repeated-measures ANOVA. A Fisher's exact test
was used for post hoc analysis. A value of P < 0.05 was considered
significant.
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RESULTS
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Effects of iNO on mRNA content of SP-A, SP-B, and SP-C.
Figure 1 demonstrates the
effects of iNO on surfactant protein mRNA content at 12 and 24 h. SP-A mRNA
content increased between 50 and 85% (P < 0.05) in the NO-treated
lambs (n = 7) at 12 h and between 60 and 77% (P < 0.05)
at 24 h, compared with pre-NO treatment (time 0) and with the
time-matched, ventilated-only lambs (n = 6). SP-B mRNA content
increased between 60 and 80% (P < 0.05) in the NO-treated lambs at
12 h and between 30 and 71% (P < 0.05) at 24 h, compared with
time 0 and with the time-matched, ventilated-only lambs. Although
there was an increase in SP-C mRNA content at both 12 and 24 h, these
differences were significant only at 12 h, increasing between 30 and 40%
(P < 0.05), compared with time 0 and with the
time-matched, ventilated-only lambs. There was no change in the SP-A, SP-B, or
SP-C mRNA content of the ventilated-only lambs at 12 and 24 h compared with
time 0 values (Fig.
2).

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Fig. 1. Effects of inhaled nitric oxide (iNO) on surfactant protein (SP)-A, SP-B,
and SP-C mRNA content at 12 and 24 h. Northern blot assays were performed from
RNA obtained from lambs before NO treatment (open bars), NO treatment for 12 h
(black bars), and 24 h (gray bars) as described in METHODS. Data
were quantified by phosphorimage analysis and normalized for the 18S signal in
each sample. Top: left 2 lanes of each blot are control
samples; middle 2 lanes are 12-h NO-treated samples; right 2
lanes are 24-h NO-treated samples. Results are expressed as the percentage of
change from pretreatment values, means ± SE. *P
< 0.05. Significant increase in mRNA content between NO-treated
lambs and the time-matched controls; P < 0.05.
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Fig. 2. Effects of ventilation alone on SP-A, SP-B, and SP-C mRNA content. Northern
blot assays were performed from RNA obtained from lambs that were ventilated
without iNO treatment at time 0 (open bars), 12 h (black bars), and
24 h (gray bars). Data were quantified by phosphorimage analysis and
normalized for the 18S signal in each sample. Top: left 2
lanes of each blot are control samples; middle 2 lanes are 12-h
NO-treated samples; right 2 lanes are 24-h NO-treated samples.
Results are expressed as the percentage of change from pretreatment values,
means ± SE. P < 0.05.
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Effects of iNO on protein levels of SP-A and SP-B.
Figure 3 demonstrates the
effects of iNO on surfactant protein content at 12 and 24 h. SP-A protein
content increased between 30 and 45% (P < 0.05) at 12 h and
decreased between 40 and 70% (P < 0.05) at 24 h, compared with
time 0 and with the time-matched, ventilated-only lambs. SP-B protein
content increased between 44 and 50% (P < 0.05) at 12 h and
decreased between 30 and 64% (P < 0.05) at 24 h, compared with
time 0 and with the time-matched ventilated-only lambs. There was no
change in SP-A or SP-B protein content of the ventilated-only sheep at 12 and
24 h compared with time 0 values
(Fig. 4).

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Fig. 3. Effects of iNO on SP-A and SP-B protein content. Quantitative dot blots
were performed on protein homogenates obtained from lambs before NO treatment
(open bars), NO treatment for 12 h (black bars), and 24 h (gray bars) as
described in METHODS. Results were normalized to total protein and
to gram of dry lung wt. Results are expressed as the percentage of change from
pretreatment values, means ± SE. *P < 0.05.
Significant increase in protein content between NO-treated lambs and
the time-matched controls; P < 0.05.
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Fig. 4. Ventilation alone did not alter SP-A and SP-B protein content. Quantitative
dot blots were performed on protein homogenates obtained from lambs that were
ventilated without iNO treatment at time 0 (open bars), 12 h (black
bars), and 24 h (gray bars). Results were normalized to total protein and to
gram of dry lung wt. Results are expressed as the percentage of change from
pretreatment values, means ± SE. P < 0.05.
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Effects of iNO on DNA content. To determine whether changes in
surfactant protein mRNAs were due to changes in cell numbers, we measured DNA
content at all time points. Figure
5 demonstrates that there was no significant change in DNA content
in the NO-treated lambs at 12 and 24 h compared with time 0 values.
There was no difference between the NO-treated lambs and their time-matched
controls. There was no difference in DNA content in ventilated-only lambs at
12 and 24 h.

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Fig. 5. Effects of iNO on DNA content. Dried DNA standards and homogenate samples
from time 0, 12 h, and 24 h were prepared and read by fluorimeter as
described in METHODS. Values were normalized to gram of dry lung
wt, and results are expressed as percent change from pretreatment values,
means ± SE. P < 0.05.
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Effects of iNO on the hemodynamic measurements. None of the
hemodynamic data was significantly different within or between groups
(Table 1).
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DISCUSSION
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Surfactant is necessary in epithelial lung fluid to decrease surface
tension at the alveolar air-liquid interface and prevent alveolar collapse at
low lung volumes (3,
7,
30). There have been many
recent in vitro and in vivo studies regarding NO and its effects on surfactant
protein homeostasis in animals. The aim of our study was to determine the
effect of inhaled NO at a moderate dose (40 ppm) on SP-A, -B, and -C gene
expression in the intact lamb.
This study demonstrates that SP-A, SP-B, and SP-C mRNA content was
significantly elevated in NO-treated lambs at 12 h and remained so for SP-A
and SP-B at 24 h. Interestingly, these findings differ from the results of
Ayad and Wong (4). In their
study, the authors demonstrate that NO exposure negatively affects SP-A gene
expression at the transcriptional level in distal respiratory epithelial cells
in vitro. There are many potential reasons for these disparate findings.
First, the model systems used in the two studies are markedly different. Ayad
and Wong conducted in vitro studies utilizing a transformed cell line, whereas
the current study was conducted in vivo utilizing a whole animal model.
Another major difference is the delivery of NO. The cells were treated with
the NO donor S-nitroso-N-acetyl penicillamine, whereas in
the present study, lambs received 40 ppm of iNO.
SP-A and SP-B protein content was increased at 12 h of iNO treatment. The
increase in surfactant protein content at 12 h could be in response to the
increase in mRNA expression at 12 h of NO treatment. Interestingly, we found
that SP-A and SP-B proteins were decreased at 24 h compared with pretreatment
levels. The decrease in surfactant proteins seen at 24 h does not appear to be
due to cell loss, as our DNA values were not significantly different in the
iNO-treated lambs at 12 and 24 h compared with time 0 values.
Although we think that the cells most likely to be injured by iNO include type
I and type II alveolar epithelial cells because they are exposed to the
highest concentration of iNO, the small increase in DNA, as well as the
increase in surfactant protein mRNA at 24 h of treatment, would argue against
type II cell injury. However, alterations in the epithelial cell surface can
be subtle (9), and if our small
increase in DNA represents an increase only in type II cells, which contribute
16% of whole lung DNA pool
(9), this might explain the
increase in surfactant mRNA seen at 24 h of therapy but not the decrease in
surfactant protein content.
It is possible that iNO resulted in oxidation or reduction of the proteins,
which may have rendered them less detectable to our antibodies. NO may have
damaged the surfactant proteins directly or by the formation of peroxynitrite
(ONOO-) and lipid peroxidation. ONOO- is an oxidant
formed rapidly by the reaction of NO and O2- in vivo.
Several in vitro studies by Haddad et al.
(13) have shown that
ONOO- inhibits pulmonary surfactant function and ability to
aggregate lipids by lipid peroxidation and damage to surfactant proteins. In
these studies, rat alveolar type II cells were exposed to an NO donor in the
presence of superoxide anion. This resulted in a 60% decrease in the rate of
surfactant synthesis, most likely due to the formation of
ONOO-.
We studied NO effects on gene expression by obtaining peripheral lung
biopsies from lambs ventilated with 21% oxygen. Oxygen use has been frequently
studied in association with NO therapy as the two are commonly used together
clinically. Hyperoxia is known to cause varying damage to surfactant proteins
and alter their function (1,
21). We chose to ventilate the
lambs with 21% oxygen to more accurately assess the sole effects of inhaled NO
on distal respiratory epithelium. Ventilation alone has been shown to alter
the pulmonary surfactant system
(29). The 13 4-wk-old lambs
were ventilated with similar support for the same length of time. There was no
difference between groups in the tidal volume, rate, or mean airway pressure
used. None of the lambs had known pulmonary abnormalities or illness. There
were no statistically significant changes in mRNA, protein, or DNA content in
the lung biopsies of the ventilated-only lambs.
In summary, iNO at 40 ppm resulted in an increase of SP-A, SP-B, and SP-C
mRNA content and SP-A and SP-B protein content at 12 h of therapy.
Interestingly, SP-A and SP-B proteins were decreased at 24 h of therapy
compared with controls despite the persistent elevation of the mRNA. The
mechanisms by which iNO affect surfactant protein gene expression remain to be
elucidated. Further in vivo studies into the mechanisms of these alterations
may lead to a better understanding of the effects of iNO on alveolar
epithelial function.
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DISCLOSURES
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Work for this project was supported in part by Robert Wood Johnson Grant
30805 (J. A. Gutierrez), National Heart, Lung, and Blood Institute Grants
HL-04372 (J. A. Gutierrez) and HL-61284 (J. R. Fineman), and American Lung
Association Grant RG-046-NL (J. A. Gutierrez).
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ACKNOWLEDGMENTS
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We acknowledge Michael Johengen, Cheri Chapin, and Wen Zhou for technical
support in this project.
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FOOTNOTES
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Address for reprint requests and other correspondence: R. B. Stuart, Dept. of
Pediatrics, Univ. of California, San Francisco, 505 Parnassus M680, Box 0106,
San Francisco, California 94143-0106 (E-mail:
rstuart{at}itsa.ucsf.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
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REFERENCES
|
---|
- Allred TF,
Mercer RR, Thomas RF, Deng H, and Auten RL. Brief 95% O2
exposure effects on surfactant protein and mRNA in rat alveolar and
bronchiolar epithelium. Am J Physiol Lung Cell Mol
Physiol 276:
L999-L1009, 1999.[Abstract/Free Full Text]
- Atz AM and
Wessel DL. Inhaled nitric oxide in the neonate with cardiac disease.
Semin Perinatol 21:
441-455, 1997.[ISI][Medline]
- Avery ME and
Mead J. Surface properties in relation to atelectasis and hyaline membrane
disease. Am J Dis Child 97:
517-523, 1959.[ISI]
- Ayad O and Wong
HR. Nitric oxide decreases surfactant protein A gene expression in H441
cells. Crit Care Med 26:
1277-1282, 1998.[ISI][Medline]
- Canadian and Network INOSGatNNR.
The neonatal inhaled nitric oxide study in the term and the near-term infant
with hypoxic respiratory failure: a multicenter randomized trial. N
Engl J Med 336:
597-604, 1997.[Abstract/Free Full Text]
- Clark RH,
Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA, Roy BJ, Keszler M,
and Kinsella JP. Low-dose nitric oxide therapy for persistent pulmonary
hypertension of the newborn. Clinical inhaled nitric oxide research group.
N Engl J Med 342:
469-474, 2000.[Abstract/Free Full Text]
- Clements JA. Dependence of pressure-volume characteristics
of lungs on intrinsic surface-active material. Am J Physiol
Dis 187: 592,
1956.
- Cuthbertson BH,
Galley HF, and Webster NR. Effect of inhaled nitric oxide on key mediators
of the inflammatory response in patients with acute lung injury.
Crit Care Med 28:
1736-1741, 2000.[ISI][Medline]
- Crapo JD,
Peters-Golden M, Marsh-Salin J, and Shelburne JS. Pathologic changes in
the lungs of oxygen-adapted rats: a morphometric analysis. Lab
Invest 39:
640-653, 1978.[ISI][Medline]
- Day RW, Lynch
JM, White KS, and Ward RM. Acute response to inhaled nitric oxide in
newborns with respiratory failure and pulmonary hypertension.
Pediatrics 98:
698-705, 1996.[Abstract]
- Fratacci MD,
Frostell CG, Chen TY, Wain JC Jr, Robinson DR, and Zapol WM. Inhaled
nitric oxide: a selective pulmonary vasodilator of heparin-protamine
vasoconstriction in sheep. Anesthesiology
75: 990-999,
1991.[ISI][Medline]
- Gutierrez JA,
Parry AJ, McMullan DM, Chapin CJ, and Fineman JR. Decreased surfactant
proteins in lambs with pulmonary hypertension secondary to increased blood
flow. Am J Physiol Lung Cell Mol Physiol
281: L1264-L1270,
2001.[Abstract/Free Full Text]
- Haddad IY, Crow
JP, Hu P, Ye Y, Beckman J, and Matalon S. Concurrent generation of nitric
oxide and superoxide damages surfactant protein A. Am J Physiol
Lung Cell Mol Physiol 267:
L242-L249, 1994.[Abstract/Free Full Text]
- Hallman M and
Bry K. Nitric oxide and lung surfactant. Semin
Perinatol 20:
173-185, 1996.[ISI][Medline]
- Hallman M, Bry
K, and Lappalainen U. A mechanism of nitric oxide-induced surfactant
dysfunction. J Appl Physiol 80:
2035-2043, 1996.[Abstract/Free Full Text]
- Hallman M,
Waffarn F, Bry K, Turbow R, Kleinman MT, Mautz WJ, Rasmussen RE, Bhalla DK,
and Phalen RF. Surfactant dysfunction after inhalation of nitric oxide.
J Appl Physiol 80:
2026-2034, 1996.[Abstract/Free Full Text]
- Kerwin JF Jr
and Heller M. The arginine-nitric oxide pathway: a target for new drugs.
Med Res Rev 14:
23-74, 1994.[ISI][Medline]
- Matalon S,
DeMarco V, Haddad IY, Myles C, Skimming JW, Schurch S, Cheng S, and Cassin
S. Inhaled nitric oxide injures the pulmonary surfactant system of lambs
in vivo. Am J Physiol Lung Cell Mol Physiol
270: L273-L280,
1996.[Abstract/Free Full Text]
- Miller OI, Tang
SF, Keech A, Pigott NB, Beller E, and Celermajer DS. Inhaled nitric oxide
and prevention of pulmonary hypertension after congenital heart surgery: a
randomised double-blind study. Lancet
356: 1464-1469,
2000.[ISI][Medline]
- Payen DM.
Inhaled nitric oxide and acute lung injury. Clin Chest
Med 21: 519-529,
ix, 2000.[ISI][Medline]
- Robbins CG,
Davis JM, Merritt TA, Amirkhanian JD, Sahgal N, Morin FC III, and Horowitz
S. Combined effects of nitric oxide and hyperoxia on surfactant function
and pulmonary inflammation. Am J Physiol Lung Cell Mol
Physiol 269:
L545-L550, 1995.[Abstract/Free Full Text]
- Roberts JD Jr,
Fineman JR, Morin FC III, Shaul PW, Rimar S, Schreiber MD, Polin RA, Zwass MS,
Zayek MM, Gross I, Heymann MA, and Zapol WM. Inhaled nitric oxide and
persistent pulmonary hypertension of the newborn. The inhaled nitric oxide
study group. N Engl J Med 336:
605-610, 1997.[Abstract/Free Full Text]
- Rossaint R,
Gerlach H, and Falke KJ. Inhalation of nitric oxide-a new approach in
severe ARDS. Eur J Anaesthesiol
11: 43-51,
1994.[ISI][Medline]
- Setaro F and
Morley CG. A modified fluorometric method for the determination of
microgram quantities of DNA from cell or tissue cultures. Anal
Biochem 71:
313-317, 1976.[ISI][Medline]
- Sison C, Bry K,
Sephus J, and Hallman M. Effects of inhaled nitric oxide and surfactant
treatment on lung function and pulmonary hemodynamics in
bronchoalveolar-lavage-induced respiratory failure. Pediatr
Pulmonol 29:
202-209, 2000.[ISI][Medline]
- Smith PK, Krohn
RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM,
Olson BJ, and Klenk DC. Measurement of protein using bicinchoninic acid.
Anal Biochem 150:
76-85, 1985.[ISI][Medline]
- Snyder SH.
Nitric oxide: first in a new class of neurotransmitters.
Science 257:
494-496, 1992.[ISI][Medline]
- Tworetzky W,
Bristow J, Moore P, Brook MM, Segal MR, Brasch RC, Hawgood S, and Fineman
JR. Inhaled nitric oxide in neonates with persistent pulmonary
hypertension. Lancet 357:
118-120, 2001.[ISI][Medline]
- Veldhuizen RA,
Tremblay LN, Govindarajan A, van Rozendaal BA, Haagsman HP, and Slutsky
AS. Pulmonary surfactant is altered during mechanical ventilation of
isolated rat lung. Crit Care Med
28: 2545-2551,
2000.[ISI][Medline]
- Weaver TE and
Whitsett JA. Structure and function of pulmonary surfactant proteins.
Semin Perinatol 12:
213-220, 1988.[ISI][Medline]