Chronic O-2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane

Abstract

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.

Full text

Chronic O
2
exposure in the newborn rat results in decreased pulmonary
arterial nitric oxide release and altered smooth muscle response to isoprostane
J. Belik, R. P. Jankov, J. Pan, M. Yi, I. Chaudhry, and A. K. Tanswell
Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8
Submitted 4 August 2003; accepted in final form 9 October 2003
Belik, J., R. P. Jankov, J. Pan, M. Yi, I. Chaudhry, and A. K.
Tanswell. Chronic O
2
exposure in the newborn rat results in de-
creased pulmonary arterial nitric oxide release and altered smooth
muscle response to isoprostane. J Appl Physiol 96: 725–730, 2004.
First published October 17, 2003; 10.1152/japplphysiol.00825.
2003.—Chronic oxygen exposure in the newborn rat results in lung
isoprostane formation, which may contribute to the pulmonary hyper-
tension evident in this animal model. The purpose of this study was to
investigate the pulmonary arterial smooth muscle responses to 8-iso-
prostaglandin F
2␣
(8-iso-PGF
2a
) in newborn rats exposed to 60% O
2
for 14 days. Because, in the adult rat, 8-iso-PGF
2␣
may have a
relaxant effect, mediated by nitric oxide (NO), we also sought to
evaluate the pulmonary arterial NO synthase (NOS) protein content
and NO release in the newborn exposed to chronic hyperoxia. Com-
pared with air-exposed control animals, 8-iso-PGF
2a
induced a sig-
nificantly greater force (P⬍0.01) and reduced (P⬍0.01) relaxation
of precontracted pulmonary arteries in the 60% O
2
-treated animals.
These changes were reproduced in control pulmonary arteries by NOS
blockade by using N
G
-nitro-L-arginine methyl ester. Pulmonary arte-
rial endothelial NOS was unaltered, but the inducible NOS protein
content was significantly decreased (P⬍0.01) in the experimental
group. Pulmonary (P⬍0.05) and aortic (P⬍0.01) tissue ex vivo NO
accumulation was significantly reduced in the 60% O
2
-treated ani-
mals. We speculate that impaired pulmonary vascular tissue NO
metabolism after chronic O
2
exposure potentiates 8-iso-PGF
2␣
-in-
duced vasoconstriction in the newborn rat, thus contributing to pul-
monary hypertension.
pulmonary hypertension; vascular; nitric oxide synthases
CHRONIC O
2
EXPOSURE INDUCES pulmonary hypertension and
pulmonary vascular remodeling in the newborn rat (14). The
mechanisms responsible for these changes are not fully under-
stood; however, recent evidence from our laboratory (10, 11)
indicates that endothelin-1, and 8-iso-prostaglandin F
2␣
(8-iso-
PGF
2␣
) may be involved in this process. Isoprostanes are
produced as a result of peroxidation of arachidonic acid by
reactive O
2
species (12). The most widely studied isoprostane
molecule is 8-iso-PGF
2␣
(12). In the adult rat pulmonary
artery, this isoprostane isomer has been shown to have dual
effects, inducing vasoconstriction in relaxed vessels and relax-
ation at lower concentrations in preconstricted vessels (13).
8-iso-PGF
2␣
-induced relaxation of vascular smooth muscle is
dependent on nitric oxide (NO) release because it can be
suppressed by a NO synthase (NOS) inhibitor (13).
Pulmonary hypertension in humans is associated with in-
creased lipid peroxidation (5), yet little is known about effects
of the isoprostanes on pulmonary vascular smooth muscle in
this condition. Induction of oxidative stress in adult rats causes
severe systemic hypertension and depressed NO availability
(23), suggesting that isoprostanes may modulate the regulation
of vascular resistance. To the best of our knowledge, an effect
of 8-iso-PGF
2␣
on pulmonary arteries in neonatal pulmonary
hypertension has not been studied. Thus the purpose of this
study was to evaluate the pulmonary arterial muscle pressor
and relaxant effect of 8-iso-PGF
2␣
in a newborn hyperoxia-
induced chronic lung injury model of pulmonary hypertension.
We have previously reported that increased pulmonary arterial
muscle contraction is present in O
2
-exposed newborn rats. We
therefore hypothesized that 8-iso-PGF
2␣
-induced force would
also be increased.
We also evaluated vascular NO production in this model.
Hyperoxia has been shown to increase lung endothelial NOS
(eNOS) expression (24) but not NO release (6) in adult rats. In
the chronically O
2
-exposed newborn rat, we have recently
documented sequestration of NO, due to peroxynitrite forma-
tion (9). Thus we hypothesized that the 8-iso-PGF
2␣
-induced
relaxation of pulmonary arterial muscle is diminished in the
newborn rat exposed to chronic hyperoxia.
METHODS
Institutional review. All procedures involving animals were con-
ducted according to criteria established by the Canadian Council for
Animal Care. Approval for the study was obtained from the Animal
Care Review Committee of the University Health Network, Toronto
Western Hospital, and Hospital for Sick Children Research Institute.
Exposure system. Pathogen-free, timed-pregnant or nonpregnant
female adult Sprague-Dawley rats (250–275 g) were obtained from
Charles River (St. Constant, Quebec, Canada). Experiments were
conducted as paired exposures with one chamber receiving 60% O
2
and the other receiving air. The animals were randomized to one or the
other chamber. O
2
and CO
2
concentrations, temperature, and humidity
were continuously monitored, recorded, and regulated by a computer
with customized software (AnaWin2 Run-Time, version 2.2.18,
Watlow-Anafaze, St. Louis, MO). Gas delivery was regulated by
customized hardware (Oxycycler model A84XOV, Biospherix, Red-
field, NY) and software (AnaWin2 Run-Time, Watlow-Anafaze), to
maintain an O
2
concentration within 0.1% of the set point. O
2
sensors
were calibrated weekly. On the anticipated day of delivery, each dam
was placed in a 80 ⫻60 ⫻50-cm plastic chamber maintained on
either air or 60% O
2
with 12:12-h light-dark cycles and the temper-
ature maintained at 25 ⫾1°C, a humidity of 50%, and a CO
2
concentration of ⬍0.5%. Equal litter sizes (10–12 pups) were main-
tained in the paired chambers. Food and water were available ad
libitum. Dams were exchanged daily between chambers to prevent
maternal O
2
toxicity. Pups were maintained in the chambers (air and
O
2
) for a 14-day exposure period. Air-treated animals served as
controls. At the termination of each exposure period, animals were
killed by an overdose of pentobarbital sodium (40 mg/kg ip).
Address for reprint requests and other correspondence: J. Belik, Univ. of
Toronto, Div. of Neonatology, Hospital for Sick Children, 555 Univ. Ave.,
Toronto, Ontario, Canada M5G 1X8 (E-mail: Jaques.Belik@SickKids.ca).
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.
J Appl Physiol 96: 725–730, 2004.
First published October 17, 2003; 10.1152/japplphysiol.00825.2003.
8750-7587/04 $5.00 Copyright ©2004 the American Physiological Societyhttp://www.jap.org 725

Organ bath studies. Fourth-generation left lung intralobar pulmo-
nary artery ring segments (average diameter ⫽100 ␮m; length ⫽2
mm) were dissected free and mounted in a wire myograph (Danish
Myo Technology). Isometric changes were digitized and recorded
online (Myodaq, Danish Myo Technology). Tissues were bathed in
Krebs-Henseleit buffer [(in mM) 115 NaCl, 25 NaHCO
3
, 1.38
NaHPO
4
, 2.51 KCl, 2.46 MgSO
4
, 1.91 CaCl
2
, and 5.56 dextrose]
bubbled with air-6% CO
2
and maintained at 37°C.
After1hofequilibration, the optimal resting tension of the tissue
was determined by repeated stimulation with 128 mM KCl until
maximum active tension was reached. All subsequent force measure-
ments were obtained at optimal resting tension.
Pulmonary vascular muscle contractile responses were normalized
to the tissue cross-sectional area as (width ⫻diameter) ⫻2 (ex-
pressed in mN/mm
2
). The relaxant response to 8-iso-PGF
2␣
was
determined by precontracting with 128 mM KCl. The thromboxane
A
2
(TxA
2
)/prostaglandin H
2
(TP) receptor blocker L-670596 (6 ⫻
10
⫺6
M) and the NOS inhibitor N
G
-nitro-L-arginine methyl ester
(L-NAME; 10
⫺5
M) were used.
Ex vivo vascular NO accumulation. NO accumulation from pul-
monary vessels and aorta was measured polarographically at 37°Cby
using a sealed water-jacketed cell fitted with a NO electrode (ISO-
NOP) and monitor (NOMKD, World Precision Instruments, Sarasota,
FL). Before each experiment, the electrode was calibrated at the same
temperature by using a standard nitrite solution (World Precision
Instruments) of known concentration, according to the manufacturer’s
instructions. Pulmonary arteries (4th generation) were dissected free
and placed in Krebs-Ringer phosphate buffer [(in mM) 130 NaCl, 5
mM KCl, 1.4 CaCl
2
, 1.3 MgSO
4
䡠7H
2
O, 10 Na
2
HPO
4
, 5 glucose, pH
7.4] equilibrated in 21% O
2
and maintained at 37°C. After a 5-min
equilibration period, NO accumulation was quantitated from the slope
of the polarograph output over a 5-min period and expressed as
nanomoles per minute. Given that the tissue could not be accurately
weighed (⬍1 mg), we measured the length and diameter of the vessel
rings and calculated their area in millimeters squared (length ⫻
diameter).
Western blot analysis. Intralobar third- to fourth-generation pulmo-
nary arterial tissue extract protein (30 ␮g) was applied to 4–20%
SDS-polyacrylamide gradient gels and electroblotted to nitrocellulose
paper. The efficiency of transfer was verified by staining the gel with
Coomassie blue after the transfer. The blot was blocked overnight
with 5% nonfat dry milk in Tween-20 Tris-base sodium (10 mM Tris
buffer, pH 7.6, 150 mM NaCl, and 0.05% Tween 20). The blots were
incubated at 4°C overnight with 1:1,000 eNOS or inducible NOS
(iNOS) (Transductions Laboratories, Lexington, KY) monoclonal
antibodies and with 1:5,000 secondary antibody for1hatroom
temperature. For eNOS, the secondary antibody was goat anti-mouse
IgG (heavy ⫹light chains)-alkaline phosphatase [(H⫹L)-AP] conju-
gate (Bio-Rad Laboratories, Hercules, CA), and for the iNOS the
antimouse IgG horseradish peroxidase (Amersham, Arlington
Heights, IL) was utilized. The cross-reactivity of the iNOS antibody
was detected by chemiluminescence by using a mixture of equal
volumes of enhanced luminol reagent and oxidizing reagent (en-
hanced chemiluminescence Western blotting analysis system, Amer-
sham). For eNOS, after washing with Tween-20 Tris-base sodium, the
blots were developed with the nitro blue tetrazolium-5-bromo-4-
chloro-3-indolyl phosphate p-toluidine salt substrate (Roche Diagnos-
tics, Laval, Quebec, Canada). Quantification was obtained by two-
dimensional scanning of the autoradiograms of the blots with a
high-resolution scanner (ImageMaster Analyser, Pharmacia, Peapack,
NJ). The results are expressed as arbitrary units (pixel intensity per
cross-sectional area of the band), as read by the scanner.
Immunohistochemistry. Lungs were removed immediately after
death, and the pulmonary vascular bed was perfused with phosphate-
buffered saline, and inflated (10 cmH
2
O pressure) and immersed in
4% paraformaldehyde in a 0.2 M sodium phosphate buffer (pH 7.4).
The tissue was postfixed and embedded in paraffin and further
processed for immunohistochemistry by using standard techniques.
Primary antibodies eNOS and iNOS (Transduction Laboratories)
were used at a dilution of 1:100 and incubated overnight at 4°C. The
secondary antibody goat anti-mouse IgG (H⫹L) biotinylated (Onco-
gene Research Products, Boston, MA) was utilized at 1:200 dilution
and incubated for 30 min at room temperature. Sections were further
incubated in avidin-biotin-peroxidase complex (ABC kit PK-4000,
Vectastain, Vector Laboratories, Burlingame, CA) for 45 min. Color
was developed by a 3,3-diaminobenzidine tetrahydrochloride sub-
strate kit (SK-4100 Vector Labs), and sections were counterstained
with Carazzi’s hematoxylin before mounting. Negative controls were
provided by primary antibody omission. Lung slices from archival
material of a newborn rat that was injected intraperitoneally with
Escherichia coli serotype 0111:B4 LPS (Sigma Chemical, St. Louis,
MO), served as a positive control for the iNOS antibody.
Drugs. L-670596 was obtained from Merck Frost (Kirkland).
8-iso-PGF
2␣
was obtained from Cayman Chemical (Ann Arbor, MI).
All other chemicals were obtained from Sigma Chemical (Oakville,
Ontario, Canada) and dissolved in Krebs-Henseleit buffer.
Sample size and data analysis. Newborn animals utilized in the
study were obtained from different litters. Four air-treated and four
60% O
2
-exposed litters were studied. The specific number or animals
utilized for each experiment is described in the figure legends.
Data were evaluated by Student’st-test or two-way ANOVA, with
multiple comparisons obtained by the Tukey-Kramer test when ap-
propriate. Statistical significance was accepted if P⬍0.05. All
statistical analysis was performed with the Number Cruncher
Statistical System (NCSS, Kaysville, UT). Data are presented as
means ⫾SE.
RESULTS
8-iso-PGF
2␣
-induced a dose-dependent increase in pulmo-
nary artery muscle force development (Fig. 1). A significant
increase in 8-iso-PGF
2␣
-induced force was found in vessels
from experimental animals (P⬍0.01), relative to controls
exposed to air (Fig. 1). In the control group, pretreatment of
vessels with the NOS inhibitor L-NAME increased the 8-iso-
Fig. 1. 8-iso-prostaglandin F
2␣
(8-iso-PGF
2␣
) pulmonary arterial muscle dose-
response curves for the control animals without (n⫽7) and with N
G
-nitro-L-
arginine methyl ester (L-NAME; n⫽4) and 60% O
2
-treated animals (n⫽4).
**P⬍0.01 by 2-way ANOVA compared with the other group values. Note
that the 8-iso-PGF
2␣
-induced force in the control group in the presence of the
nitric oxide (NO) inhibitor (L-NAME) increased to values comparable to the
60% O
2
-treated group.
726 CHRONIC O
2
EXPOSURE AND NEWBORN LUNG SMOOTH MUSCLE
J Appl Physiol •VOL 96 •FEBRUARY 2004 •www.jap.org

PGF
2␣
-induced force to levels comparable to the experimental
group (Fig. 1).
To test for the 8-iso-PGF
2␣
-induced relaxation response,
pulmonary arteries were prestimulated with KCl (128 mM). In
control arteries, 8-iso-PGF
2␣
-induced relaxation was observed
only at the highest concentration tested (10
⫺6
M). However, in
the presence of L-670596, a TxA
2
receptor blocker, a signifi-
cant (P⬍0.01) dose-dependent relaxation was seen at all
concentrations, which was suppressed by the addition of L-
NAME (Fig. 2). When 8-iso-PGF
2␣
-induced relaxation of
air-exposed control and 60% O
2
-exposed experimental arteries
were compared in the presence of TP-receptor blockade, the
arteries from experimental animals showed a significantly
decreased (P⬍0.01) relaxant response (Fig. 3).
Pulmonary artery and aorta from control and experimental
animals were examined for basal NO accumulation (Fig. 4).
Compared with air controls, NO accumulation was signifi-
cantly decreased in the pulmonary arteries (P⬍0.05) and
aortas (P⬍0.01) of the experimental animals (Fig. 4). The
group differences were not related to the amount of tissue
present in the polarographic chamber, because the pulmonary
arterial and aorta tissue area for the control (5.5 ⫾0.4 and
7.1 ⫾0.4 mm
2
, respectively) and experimental (5.8 ⫾1.2 and
10.2 ⫾1.8 mm
2
, respectively) groups were not statistically
different.
Finally, we evaluated pulmonary arterial NOS protein con-
tent (Figs. 5 and 6). As shown in Fig. 5 by Western blot and
Fig. 6 by immunohistochemistry, we found no differences in
eNOS content between control and experimental groups. The
tissue iNOS protein content was significantly reduced (P⬍
0.01) in the 60% O
2
-exposed animals (Fig. 5) but was below
detection limits by immunohistochemistry in the lungs of both
groups (Fig. 6).
DISCUSSION
Exposure of newborn rats to 60% O
2
for 14 days results in
pulmonary vascular remodeling and changes in pulmonary
arterial smooth muscle contractile properties (2, 8). Our group
has recently reported (9) that enhanced reactive O
2
species-
mediated inactivation and sequestration of NO, attributed to
peroxynitrite formation, is present in the lungs of hyperoxia-
exposed newborn rats on the basis of increased lung nitroty-
rosine formation. In this study, together with a reduced pul-
monary vascular iNOS protein content, we have confirmed that
NO release is significantly decreased in experimental animals.
This finding, which indicates decreased functional release of
NO, may well account for alterations in pulmonary arterial
smooth muscle response to 8-iso-PGF
2␣
in hyperoxia-exposed
animals.
8-iso-PGF
2␣
has a dual effect on the pulmonary arterial
muscle of the adult rats: it may cause either contraction of
relaxed vessels or relaxation of precontracted vessels in a
Fig. 2. 8-iso-PGF
2␣
dose-response curves for the control animals’pulmonary
arterial muscles precontracted with 128 mM KCl in the absence (n⫽7) and
presence of thromboxane A
2
/prostaglandin H
2
(TP) receptor (L-670596)
blockade [TP(⫺); n⫽4] as well TP receptor and NO synthase (NOS)
blockade [TP(⫺)⫹L-NAME; n⫽4]. **P⬍0.01 by 2-way ANOVA
compared with other group values. The KCl-induced stress was 2.3 ⫾0.1,
1.9 ⫾0.1, and 2.2 ⫾0.1 mN/mm
2
in the control, TP(⫺), and TP(⫺)⫹
L-NAME groups, respectively. Note that the 8-iso-PGF
2␣
-induced relaxation is
unmasked after TP-receptor blockade.
Fig. 3. 8-iso-PGF
2␣
-induced relaxation for the control (n⫽7) and 60%
O
2
-treated (n⫽4) animals’pulmonary arterial muscles precontracted with 128
mM KCl in the presence of TP-receptor blocker (L-670596). The KCl-induced
stress was 2.3 ⫾0.1 and 4.1 ⫾0.2 mN/mm
2
in the control and 60% O
2
-treated
groups, respectively. Note that the 8-iso-PGF
2␣
-induced relaxation is signifi-
cantly decreased (**P⬍0.01) in the O
2
-treated animals.
Fig. 4. NO release from control and 60% O
2
-treated pulmonary arterial (n⫽
4 and n⫽6, respectively) and aortic (n⫽4 and n⫽9, respectively) tissue.
**P⬍0.01, *P⬍0.05 compared with control values. Note that pulmonary
arterial and aorta tissue NO releases are significantly reduced in the experi-
mental animals.
727CHRONIC O
2
EXPOSURE AND NEWBORN LUNG SMOOTH MUSCLE
J Appl Physiol •VOL 96 •FEBRUARY 2004 •www.jap.org

concentration-dependent manner. The relaxant response to
8-iso-PGF
2␣
in the presence of TP-receptor blockade is con-
sistent with there being two receptors for this isoprostane (7).
The TP receptor is localized on the smooth muscle membrane
and is involved in the contraction response. The other receptor
has not been characterized, but it is believed to be located on
the endothelial cell membrane and responsible for the 8-iso-
PGF
2␣
-induced relaxation. Our laboratory has previously
Fig. 5. Western blot analysis of pulmonary
arterial tissue from control (n⫽4) and 60%
O
2
-treated animals (n⫽4) for endothelial
NOS (eNOS) and inducible NOS (iNOS).
Although no change in eNOS protein con-
tent was observed, 60% O
2
treatment re-
sulted in a significant decrease (**P⬍0.01)
in iNOS protein content.
Fig. 6. Representative pulmonary arterial immunohistochemis-
try for eNOS and iNOS. No obvious group staining difference
was noted for eNOS. iNOS immunostaining was absent in the
control (C) and 60% O
2
-treated groups but present in the
positive-control (⫹control) sample of a pulmonary arterial
vessel of an Escherichia coli LPS-treated newborn rat. Magni-
fication ⫽⫻100, ⫻200, and ⫻400, as illustrated.
728 CHRONIC O
2
EXPOSURE AND NEWBORN LUNG SMOOTH MUSCLE
J Appl Physiol •VOL 96 •FEBRUARY 2004 •www.jap.org

shown (3) that the newborn pulmonary vascular muscle re-
sponse to 8-iso-PGF
2␣
is clearly distinct from the adult. In the
newborn, 8-iso-PGF
2␣
induces less muscle contraction than the
adult, and its relaxant effect is only evident after TP-receptor
blockade.
In this study, we have shown that blockade of NOS with
L-NAME resulted in increased 8-iso-PGF
2␣
-induced force and
decreased relaxation of precontracted control pulmonary arter-
ies. These changes mirror the pattern observed in pulmonary
arterial tissue of chronic hyperoxia-exposed animals. Such
evidence, in conjunction with the documented reduced NO
release from pulmonary arteries of experimental animals, sug-
gests that the altered response to 8-iso-PGF
2␣
was caused by an
effect of chronic hyperoxia on the pulmonary vascular NO
metabolism.
Conflicting data exist concerning the effect of chronic hy-
peroxia on NO production in the adult rat. A ⬍72-h exposure
to 85% O
2
in adult rats increased lung iNOS but not eNOS
content without an effect on exhaled gas NO content (6).
Exposure to 85% O
2
for 28 days resulted in increased lung
vascular tissue eNOS content and activity (21), whereas 95%
O
2
exposure for up to 5 days caused a decrease in eNOS
activity (1) in adult rats. Isolated and perfused adult lungs
exposed to 90% O
2
for 48 h showed loss of the hypoxic
pulmonary vasoconstrictor response, which was interpreted
as being related to upregulation of NO production by the
lung (22).
In 3-day-old rats exposed to 95% O
2
for up to 14 days,
eNOS activity was decreased, but the lung tissue protein
content increased, whereas iNOS activity increased and its
protein content decreased, compared with age-matched con-
trols (18). Ours is only the second study to address the effect on
chronic hyperoxia on the pulmonary vascular tissue NOS
content of newborn rats. It contrasts with the former study in
subjecting the newborn rats to 60% O
2
for 14 days starting
from birth. As mentioned previously, no changes in eNOS
protein content or lung immunohistochemical staining were
noted, but iNOS protein content was significantly reduced in
experimental animals’pulmonary vascular tissue in the present
study, in keeping with the findings of Radomsky et al. (18).
The mechanism accounting for the decrease in the pulmonary
artery iNOS content is presently unclear. The apparent discrep-
ancy between the absent immunostaining but positive Western
results for iNOS likely reflects the low tissue iNOS protein
content, which makes detection by immunohistochemistry
technically difficult. It is clear that the antibody utilized in this
study can recognize iNOS in paraffin-preserved tissue, because
positive immunostain was present in the LPS-stimulated pos-
itive control newborn lung. Although not measured in this
study, the pulmonary arterial eNOS activity and phosphoryla-
tion in the 60% O
2
-exposed newborns is likely normal given
our previously reported data (2) in this model relative to
unaltered acetylcholine-induced relaxation in these vessels.
Last, the aorta NO release in the experimental animals was
significantly lower than the control tissue, which suggests that
chronic hyperoxia has a similar effect on systemic vascular NO
metabolism, as observed in the pulmonary vessels. This finding
is not surprising given that endothelial-derived NO is reduced
in vascular diseases such as systemic hypertension in humans
(17) and animal models of atherosclerosis (20). Furthermore,
induction of chronic oxidative stress by inhibition of glutathi-
one synthase induces systemic hypertension in adult rats via
a mechanism involving NO inactivation by reactive O
2
spe-
cies (24).
The novel data reported in this study may be relevant to our
understanding of the pathogenesis of pulmonary hypertension
in the human neonate. Pulmonary disease-induced lipid per-
oxidation (4, 15, 16) and the therapeutic use of supplemental
O
2
(19) result in isoprostane production in the lung. At low
concentrations, isoprostanes may have a physiological role in
the lung by promoting pulmonary vasodilation via NO release
(12). Yet, in pulmonary hypertension, where an altered vascu-
lar NO metabolism is present, increased 8-iso-PGF
2␣
and
possibly other isoprostane production will promote pulmonary
vascular muscle contraction, contributing to the maintenance
of a high pulmonary vascular resistance in this disease.
In conclusion, we have shown that chronic exposure to 60%
O
2
in newborn rats results in decreased pulmonary vascular
tissue NO release. This finding likely accounts for the observed
enhanced pulmonary arterial muscle contraction and reduced
relaxation in response to 8-iso-PGF
2␣
. Considering that the
lung levels of isoprostane are markedly increased in experi-
mental animals exposed to 60% O
2
, these changes are likely to
play a major role in the pathogenesis of pulmonary hyperten-
sion in this animal model.
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