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Endogenous Nitric Oxide Can Act as Beneficial or Deleterious in the Hypoxic Lung Depending on the Reoxygenation Time.

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THE ANATOMICAL RECORD 293:2193–2201 (2010)
Endogenous Nitric Oxide Can Act as
Beneficial or Deleterious in the Hypoxic
Lung Depending on the
Reoxygenation Time
ALMA RUS, FRANCISCO MOLINA, M. ÁNGELES PEINADO,
AND M. LUISA DEL MORAL*
Department of Experimental Biology, University of Jaén, Jaén, Spain
ABSTRACT
Nitric oxide (NO) has been implicated in many pathophysiological situations in the lung, including hypoxia/reoxygenation. This work seeks to
clarify the current controversy concerning the double protective/toxic role
of endogenous NO under hypoxia/reoxygenation situations in the lung by
using a nitric oxide synthase (NOS) inhibitor, in a novel approach to
address the problems raised from assaults under such circumstances.
A follow-up study was conducted in Wistar rats submitted to hypoxia/
reoxygenation (hypoxia for 30 min; reoxygenation of 0 h, 48 h, and 5
days), with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM, in drinking water). Lipid peroxidation, apoptosis
level, protein nitration, in situ NOS activity and NO production (NOx)
were analyzed. This is the first work to focus on the time-course effects of
L-NAME in the adult rat lung submitted to hypoxia/reoxygenation. The
results showed that after L-NAME administration, in situ NOS activity
was almost completely eliminated and consequently, NOx levels fell. Lipid
peroxidation and the percentage of apoptotic cells rose at the earliest
reoxygenation time (0 h), but decreased in the later period (48 h and 5
days). Also nitrated protein expression decreased at 48 h and 5 days posthypoxia. These results suggest that NOS-derived NO exerts two different
effects on lung hypoxia/reoxygenation injury depending on the reoxygenation time: NO has a beneficial role just after the hypoxic stimulus and a
deleterious effect in the later reoxygenation times. Moreover, we propose
that this dual role of NO depends directly on the producer NOS isoform.
C 2010 Wiley-Liss, Inc.
Anat Rec, 293:2193–2201, 2010. V
Key words: hypoxia; nitric oxide; nitric oxide synthase; L-NAME;
apoptosis; lipid peroxidation; reoxygenation
INTRODUCTION
Hypoxia is one of the most frequently encountered
types of stress in health and disease. To find useful remedies that are capable of ameliorating its casualty is an
essential effort. Although the underlying mechanisms of
the hypoxia-induced injury and cell death are still not
fully understood, it has been shown that hypoxia induces
nitric oxide (NO) overproduction (Kiang and Tsen, 2006).
In the lungs, endogenous NO plays an important role in
regulating a number of physiological and pathological
processes, including hypoxia. NO is produced by three
isoenzymes of nitric oxide synthase (NOS): endothelial
C 2010 WILEY-LISS, INC.
V
(eNOS or NOS III), inducible (iNOS or NOS II), and
neuronal (nNOS or NOS I) (Vaughan et al., 2003).
Grant sponsor: Ministry of Education and Sciences of Spain;
Grant numbers: SAF-2003-04398-C02-02, BIO 2000-0405-P4-05.
*Correspondence to: M. Luisa del Moral, Department of Experimental Biology, University of Jaén, Jaén, Spain. Fax: þ34953-211875. E-mail: mlmoral@ujaen.es
Received 3 March 2010; Accepted 1 June 2010
DOI 10.1002/ar.21229
Published online 23 August 2010 in Wiley Online Library
(wileyonlinelibrary.com).
2194
RUS ET AL.
Whereas eNOS and nNOS are mostly calcium/calmodulin-dependent and usually express constitutively (Palmer
et al., 1987), iNOS is typically independent of intracellular calcium concentration and can be induced in a wide
variety of cell types in response to some stimuli, such as
hypoxia (Jung et al., 2000; Ricciardolo et al., 2004), or
after stimulation with inflammatory mediators (Schulz
et al., 1991; Kelly et al., 1996). Two main sites of NO
production have been identified in the lung, the vasculature and the airways (Kobzik et al., 1993; Sherman
et al., 1999). The main isoform in vascular endothelium
is eNOS. In the airways, all three isoforms have been
detected in the bronchial and bronchiolar epithelium
(Kobzik et al., 1993; Shaul et al., 1995), iNOS being the
major airway epithelial isoform (Guo et al., 1995).
The reoxygenation of the hypoxic tissues is characterized by the formation of both reactive oxygen species
(ROS) and reactive nitrogen species (RNS), resulting in
widespread lipid peroxidation, protein oxidative and
nitrosative modifications, alterations in DNA (Szabo,
1996) as well as apoptosis and necrosis (McCord et al.,
1985). Peroxynitrite, formed by the reaction of NO and
superoxide, is a ROS and RNS that can alter protein
function by nitrating phenolic rings, including tyrosine,
to create nitrotyrosines (Haddad et al., 1994).
A number of works in the literature have shown controversial results reporting that NO can act both as a
destructive as well as a protective agent in the pathogenesis of the hypoxia/reoxygenation injuries in the lung.
In this sense, while several investigations have
reported that NOS inhibitors exacerbate hypoxic injury
in the lung (Naoki et al., 1999; Shirai et al., 2003),
other studies using pharmacological inhibition of NOS
showed protective effects against the same type of injuries (Baber et al., 2005; Barer et al., 2006). All this
controversy exemplifies the need to provide a clearer
view of the role of NOS-derived NO under hypoxia/
reoxygenation situations in the lung. For this, we studied parameters of cell and tissue damage (lipid peroxidation, apoptosis, and levels of nitrated proteins)
before and after treatment with the NOS inhibitor NGnitro-L-arginine methyl ester (L-NAME) in the lung of
Wistar rats submitted to hypoxia/reoxygenation. L-NAME
is an analogue of L-arginine that acts as a nonselective inhibitor of the activity, but not of the expression, of the
three NOS (Rees et al., 1990). Moreover, it bears mentioning that the literature dealing with this inhibitor in
hypoxic lungs is very scant, and thus its possible effects
on this organ remain completely unknown. This is the
first available study that describes the time-course effects
of this NOS inhibitor in the adult rat lung submitted to
hypoxia/reoxygenation.
METHODS
Animals
The study was performed on mature adult (4–5
months old) male albino Wistar rats kept under standard conditions of light and temperature and allowed ad
libitum access to food and water. All the experiments
were conducted according to E.U. guidelines on the use
of animals for biochemical research (86/609/EU).
Experimental Protocol and NOS Inhibitor
Administration
The acute hypobaric hypoxia was carried out as previously published by our group (Lopez-Ramos et al., 2005;
Martı́nez-Romero et al., 2006; Rus et al., 2010a). Briefly,
animals were placed in a chamber in which the air pressure was controlled by means of a continuous vacuum
pump and an adjustable inflow valve. Hypoxia was
induced by downregulating the environmental O2 pressure to a final barometric pressure of 225 mmHg, resulting in a 48 mmHg O2 partial pressure (pO2). These
conditions were maintained for 30 min. The ascent and
descent speed was maintained below 1,000 feet/min.
After the hypoxia period, animals were kept under normobaric normoxic conditions for different reoxygenation
times (0, 48 h, and 5 days), and then were sacrificed.
Control animals were maintained for 30 min in the
chamber under normobaric normoxic conditions, before
being sacrificed.
The nonselective NOS inhibitor L-NAME or NG-nitroL-arginine methyl ester (Sigma) (1.5 mM) was dissolved
in the drinking water of the animals for 2 weeks before
hypoxia (Alonso et al., 2002). After the hypoxia period,
animals were kept under normobaric normoxic conditions for different reoxygenation times (0, 48 h, and 5
days), and then were sacrificed. Control animals were
maintained for 30 min in the chamber under normobaric
normoxic conditions, before being sacrificed. The following experimental groups (n ¼ 5 rats per group) were
studied:
1. Control: rats maintained for 30 min in the chamber
under normobaric normoxic conditions.
2. Hypoxia/reoxygenation: rats submitted to 30 min
of hypoxia followed by 0 h, 48 h, and 5 days of
reoxygenation.
3. Control þ L-NAME: control rats treated with
L-NAME.
4. Hypoxia/reoxygenation þ L-NAME: rats submitted to
the same procedure as the second group but treated
with L-NAME.
A total of 40 albino Wistar rats were used for the biochemical experiments (five animals per experimental
group). After the corresponding reoxygenation times, the
rats were killed by cervical dislocation and the lungs
were immediately removed, rinsed in saline solution,
and stored at 80 C until used. Another 40 rats were
used for histochemistry and immunohistochemistry (five
animals per experimental group). The rats were anaesthetized with Ketolar (Parke Davis, 1 mL/250 g weight)
by intraperitoneal injection and perfused at each reoxygenation time. Then, the lungs were removed, rinsed in
saline solution, and fixed.
NADPH-Diaphorase Histochemistry
NOS have been shown to have NADPH-diaphorase activity, as evidenced by colocalization and coprecipitation
of NADPH-diaphorase and NOS activity (Snyder, 1992).
In fact, NADPH-diaphorase histochemistry has been
widely used as an indirect way to determine in situ NOS
activity (Kugler et al., 1994; Roufail et al., 1995; Moraes
et al., 2001). Lung sections, 40-lm thick, were cut using
DUAL ROLES OF NO IN THE HYPOXIC LUNG
a cryostat (2800 Frigocut E, Reichert-Jung Vienna, Austria). Free-floating sections were incubated for 4 h in
PBS containing 0.1% Triton X-100. After several washes
in 0.1 M Tris-HCl pH 7.4 buffer, they were incubated in
the dark for 45 min at 37 C in 0.1 M Tris-HCl pH 7.4
containing 1 mM b-NADPH and 2 mM NBT (in 70%
dimethylformamide). The sections were then washed
twice with 0.1 M Tris-HCl pH 7.4, quickly dehydrated in
a graded ethanol series, cleared, and mounted in DPX
(Fluka, Madrid, Spain).
NO Measurement
The reaction of NO with ozone results in the emission
of light, and this light (emitted in proportion to the NO
concentration) is the basis for one of the most accurate
NO assays available (Fontijn et al., 1997; Laitinen et al.,
1993). NO production was indirectly quantified by measuring nitrate/nitrite and S-nitrose compounds (NOx)
using an ozone chemiluminescence-based method. For
this technique, lungs were homogenized in PBS with
protease inhibitors. Homogenates were then sonicated,
centrifuged, and deproteinized with NaOH 0.8 N and
ZnSO4 16% solutions. The total amount of NOx was
determined by a modification (Lopez-Ramos et al., 2005)
of the procedure described by Braman and Hendrix
(1989) using a NO analyzer (NOATM 280i Sievers Instruments). A saturated solution of vanadium chloride (VCl3)
in 1 M HCl was added to the nitrogen-bubbled purge
vessel fitted with a cold water condenser and a water
jacket to heat the reagent to 90 C using a circulating
bath. HCl vapors were removed by a gas bubbler containing 1 M NaOH. The gas-flow rate into the detector
was controlled by a needle valve adjusted to yield a constant pressure. Once the detector signal was stabilized,
samples were injected into the purge vessel to react with
the reagent, converting NOx to NO, which was then
detected by ozone-induced chemiluminescence. NOx concentrations were calculated by comparison with standard solutions of sodium nitrate. Final NOx values were
referred to the total protein concentration in the initial
extracts.
Thiobarbituric Acid Reactive Substances
Thiobarbituric acid reactive substances (TBARS) were
determined in lung homogenates as described by Buege
and Aust (1978). Briefly, lungs were homogenized in
PBS, and then sonicated and centrifuged. In the supernatant, the amount of proteins was determined using
the Bradford assay (1976). After 700 lL of thiobarbituric
acid reagent (15% TCA or trichloroacetic acid, 0.38%
TBA or 2 thiobarbituric acid and 2% HCl or chloride
acid) was added to 300 lL of the supernatant, the solution was heated at 95 C for 15 min. After heating, the
tubes were cooled in a water bath and centrifuged. The
absorbance of the supernatant was read at 535 nm.
TUNEL Assay for Assessment of Apoptotic
Cell Death and Image Processing
Terminal deoxynucleotidyl transferase (TdT)-mediated
desoxyuridinetriphosphate (dUTP) nick end-labelling
(TUNEL) is a technique to estimate apoptosis in tissue
sections. The protocol was performed in sections
2195
obtained from lungs embedded in paraffin according to
the manufacturer’s recommendations (TdT-FragELTM
DNA Fragmentation Detection Kit, Calbiochem). Deionized water was replaced by TdT enzyme as a negative
control. Apoptotic bodies were stained brown.
Ten similar microphotographs per rat were digitally
captured with a light microscope (Olympus, Hamburg,
Germany). They were then analyzed, after background
subtraction (minimal particle size 10 pixels), in two different color channels using ImageJ (an NIH image analysis and processing software downloaded free from
http://rsbweb.nih.gov/ij/). The image derived from the
green channel was used to determine the number of living cells while the image acquired from the red one was
used to determine the apoptotic cells. The percentage of
area of apoptotic cells in each microphotograph was
quantified by computer-assisted image analysis using
the same software.
Western Blot Analysis for Nitrotyrosine
Expression
For Western blot analysis, equal amounts of denatured
lung total-protein extracts were loaded and separated in
7.5% SDS-polyacrylamide gel. Proteins in the gel were
transferred to a PVDF membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) and then blocked.
Polyclonal rabbit anti-3-nitro-L-tyrosine A4 antibody
(1:3000, gift from Professor J. Rodrigo from CSIC Cajal
Institute of Madrid) was used to detect nitrated proteins,
and monoclonal antibody to a-tubulin (Sigma) was used
as the internal control. Antibody reaction was revealed
with chemiluminescence detection following the manufacturer’s recommendations (ECL kit, Amersham Corp.,
Buckinghamshire, UK).
Nitrotyrosine Immunohistochemistry
Lung samples were embedded in paraffin (Paraplast
Extra, Tyco). Sections were incubated with 10% goat serum during 30 min. Afterward, they were first incubated
with diluted polyclonal rabbit anti-3-nitro-L-tyrosine A4
antibody (1:500, gift from Professor J. Rodrigo from
CSIC Cajal Institute of Madrid), used to detect nitrated
proteins, in PBS overnight at 4 C, and later with a goat
anti-rabbit biotinylated secondary antibody (Pierce) followed by peroxidase-linked ABC. The peroxidase activity
was demonstrated following the nickel-enhanced diamino-benzidine procedure (Shu et al., 1988). Sections
were mounted on slides, dehydrated, and covered using
DPX. Controls for background staining were performed
by replacing the primary antibody with PBS.
Statistical Analysis
Data were expressed as mean standard deviation
(SD). The statistical treatment to evaluate significant
differences between groups was performed with SPSS
15.0 software. The data followed a normal distribution
(tested with Kolmogorov–Smirnov test) and the principle
of homoscedasticity of variances (tested with Levene
test), and were tested by a two-way ANOVA. The statistical significance was established by applying an
unpaired Student’s t-test to compare differences between
means. The statistically significant differences regarding
2196
RUS ET AL.
Fig. 2. Influence of hypoxia/reoxygenation on nitrate, nitrite, and
other nitrose compounds (NOx) in rat lung (lmol/mg protein). Experimental groups: Control and 0 h, 48 h, and 5 days posthypoxia; control
L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days L-NAME posthypoxia. Results are mean values of three independent experiments with
five animals per group. The statistically significant differences regarding the control group in the untreated groups were expressed as aP <
0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically significant differences between the treated groups and the corresponding
untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01;
and zP < 0.001.
Fig. 1. NADPH-diaphorase staining. (A–H): Micrographs showing in
situ NOS activity in rat lung sections in endothelial cells (arrows) and
bronchiolar epithelial cells (arrow heads). Experimental groups: Control
(C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME
and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia.
Micrographs (C), (E), and (G) show the presence of slightly more
intense NADPH-diaphorase staining in both types of cells when compared with the control group in the untreated groups. Micrographs (B),
(D), (F), and (H) do not show NADPH-diaphorase staining in relation to
the untreated groups. Scale bars ¼ 200 lm.
the control group in the untreated groups were
expressed as aP < 0.05; bP < 0.02; cP < 0.01; and dP <
0.001. The statistically significant differences between
the treated groups and the corresponding untreated
groups were expressed as wP < 0.05; xP < 0.02; yP <
0.01; and zP < 0.001.
RESULTS
NADPH-Diaphorase Histochemistry
In the microphotographs (Fig. 1, microphotographs A,
C, E, and G taken from Rus et al., 2010b), the NADPHdiaphorase staining, representative of in situ NOS activity, was found in all the experimental groups in endothelial cells and bronchiolar epithelial cells. An increase in
NADPH-diaphorase staining was detected at 0 h, 48 h,
and 5 days posthypoxia in those cell types in the
untreated groups. On the other hand, after the treat-
ment with the nonselective NOS inhibitor L-NAME, the
NADPH-diaphorase staining was almost completely
eliminated in all the experimental groups.
NO Production
Figure 2 shows the determinations of nitrate/nitrite
and other S-nitrose compounds (NOx) in the different
experimental groups. NOx levels rose early, at 0 h of
reoxygenation (P < 0.001), in the untreated groups.
However, the administration of this NOS inhibitor significantly lowered NOx levels in control (P < 0.001) and
throughout all the reoxygenation periods: 0 h (P <
0.001), 48 h (P < 0.05), and 5 days (P < 0.05).
Thiobarbituric Acid Reactive Substances
Malondialdehyde and other lipid peroxidation products, which react with thiobarbituric acid, are good indicators of oxidative stress (Drapper et al., 1993). Figure 3
shows that hypoxia/reoxygenation raised the TBARS
level at 48 h (P < 0.001), and 5 days (P < 0.001) posthypoxia in the untreated groups. Moreover, in the groups
treated with L-NAME, TBARS increased at 0 h (P <
0.05), but decreased in the later reoxygenation periods
(48 h: P < 0.02; 5 days: P < 0.001).
TUNEL Assay
The TUNEL assay, which identifies apoptotic cells,
showed that hypoxia/reoxygenation significantly raised
DUAL ROLES OF NO IN THE HYPOXIC LUNG
2197
Fig. 3. Influence of hypoxia/reoxygenation on lipid peroxidation in
rat lung. Results were expressed as absorbance at 535 nm. Experimental groups: control and 0 h, 48 h, and 5 days posthypoxia; control
L-NAME and 0 h L-NAME, 48 h L-NAME, and 5 days L-NAME posthypoxia. Results are mean values of three independent experiments with
five animals per group. The statistically significant differences regarding the control group in the untreated groups were expressed as aP <
0.05; bP < 0.02; cP < 0.01; and dP < 0.001. The statistically significant differences between the treated groups and the corresponding
untreated groups were expressed as wP < 0.05; xP < 0.02; yP < 0.01;
and zP < 0.001.
TABLE 1. Quantitative data from image
analysis of histological sections of rat lungs
stained for TUNEL assay
Group
Untreated groups
Control
0h
48 h
5 days
L-NAME-treated groups
Control
0h
48 h
5 days
Percentage of area of
apoptotic cells (%)
3.31
3.80
20.18
34.67
1.10
1.00
2.52d
2.47d
5.12
22.84
9.46
7.13
2.07
1.71z
2.20z
2.63z
Experimental groups: control and 0 h, 48 h, and 5 days
posthypoxia; control L-NAME and 0 h L-NAME, 48 h LNAME, and 5 days L-NAME posthypoxia. Results are mean
values of 50 microphotographs (10 microphotographs per
animal and five animals per group) SD. The statistically
significant differences regarding the control group in the
untreated groups were expressed as aP < 0.05; bP < 0.02;
c
P < 0.01.
d
P < 0.001.
The statistically significant differences between the treated
groups and the corresponding untreated groups were
expressed as wP < 0.05; xP < 0.02; yP < 0.01; and.
z
P < 0.001.
Fig. 4. (A–H): Micrographs showing histological sections of rat lung
stained for TUNEL assay. Experimental groups: control (C) and 0 h, 48
h, and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME,
48 h L-NAME, and 5 days (5d) L-NAME posthypoxia. Apoptotic,
TUNEL-positive cells are indicated by the brown nuclear staining
(arrows). Micrographs (E) and (G) show higher apoptosis levels when
compared with the control group in the untreated groups. Micrograph
(D) shows higher apoptosis levels in relation to the corresponding
untreated group. Micrographs (F) and (H) show lower apoptosis levels
in relation to the corresponding untreated groups. Scale bars ¼ 50
lm.
the percentage of apoptotic cells at the late posthypoxia
times (48 h: P < 0.001, 5 days: P < 0.001) in the
untreated groups (Table 1). However, after the treatment
with L-NAME, this percentage rose at the earliest reoxygenation period (0 h: P < 0.001), but decreased in the
later posthypoxia times (48 h: P < 0.001, 5 days: P <
0.001) when compared with the corresponding untreated
groups (Fig. 4, microphotographs A, C, E, and G taken
from Rus et al., 2010b).
Nitrotyrosine Expression
Three nitrotyrosine immunoreactive bands, corresponding to proteins of 126, 112, and 72 kDa, were
detected in all the experimental groups (Fig. 5, right
panel). The quantitative evaluation of the bulk-nitrated
proteins increased significantly from 48 h (P < 0.001) to
5 days (P < 0.001) posthypoxia in the untreated groups
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RUS ET AL.
Fig. 5. Influence of hypoxia/reoxygenation on nitrotyrosine-modified
protein expression in rat lung. Left panel: densitometric quantification
of nitrotyrosine-modified proteins in the experimental groups: Control
(C) and 0 h, 48 h, and 5 days (5d) posthypoxia; control (C) L-NAME
and 0 h L-NAME, 48 h L-NAME, and 5 days (5d) L-NAME posthypoxia.
Results were expressed as arbitrary units (A.U.). Results are mean values
of three independent experiments with five animals per group. Right
panel: representative autoradiography of the nitrotyrosine-modified protein bands; a-tubulin immunodetection was also included as a proteinloading control. The statistically significant differences regarding the control group in the untreated groups were expressed as aP < 0.05; bP <
0.02; cP < 0.01; and dP < 0.001. The statistically significant differences
between the treated groups and the corresponding untreated groups
were expressed as wP < 0.05; xP < 0.02; yP < 0.01; and zP < 0.001.
(Fig. 5, left panel). However, the administration of this
nonselective inhibitor decreased nitrated protein expression at 48 h (P < 0.05), and 5 days (P < 0.02) of
reoxygenation.
nation situations in the lung. The results indicate that
our model of hypoxia/reoxygenation increased the
NADPH-diaphorase staining, indicative of in situ NOS
activity, in endothelial and bronchiolar epithelial cells
throughout the reoxygenation periods (0 h, 48 h, and 5
days) in the untreated groups. However, this staining
was almost completely eliminated in those cell types after L-NAME administration, as expected. On the other
hand, we have also shown that NO levels, indirectly
quantified by nitrate/nitrite and S-nitrose compounds
(NOx), significantly rose at 0 h posthypoxia in the
untreated groups when compared with the normobaric
normoxic control. Nonetheless, the administration of this
nonselective NOS inhibitor significantly lowered NOx
levels in all the experimental groups. Because it is well
known that L-NAME is a NOS inhibitor (Viñas et al.,
2006; Bertuglia, 2008), the decrease reported here in
NOx levels and in situ NOS activity should be attributed
to the inhibition of NOS isoforms.
Our model of hypoxia/reoxygenation significantly raised
the lipid peroxidation level, determined by TBARS, at
48 h, and 5 days posthypoxia in the untreated groups,
proposing that changes consistent with oxidative processes occur in the lung in response to hypoxia/reoxygenation. On the other hand, once L-NAME was
administrated, the TBARS levels significantly increased
during the reoxygenation period at 0 h, but decreased in
the later posthypoxia times (48 h, 5 days). Our results
have also shown that our hypoxia/reoxygenation model
provokes cell damage, indicated by the use of the
TUNEL assay, which measures DNA damage and is an
indicator of apoptosis. In this sense, an increased percentage of apoptotic cells were detected at 48 h and 5
days posthypoxia in the untreated groups. Nevertheless,
the L-NAME treatment produced similar effects on
Nitrotyrosine Immunohistochemistry
In all the experimental groups, the nitrotyrosine immunoreactivity was detected in bronchiolar epithelial
cells and vascular endothelial cells (Fig. 6, microphotographs A, C, E, and G taken from Rus et al., 2010b).
Corroborating the previous results, the nitrotyrosinepositive staining was detected more intense from 48 h to
5 days posthypoxia in these cell types in the untreated
groups and less intense during those times in the groups
treated with the NOS inhibitor L-NAME.
DISCUSSION
Hypoxia-associated pathophysiology is complicated and
elusive but noticeably significant. Many adverse effects of
hypoxia are commonly observed under conditions generated by ischemia. Like reperfusion, reoxygenation eventually does not completely reverse the hypoxia-induced
changes. The complexity of the cell response to hypoxia
complicates efforts to design approaches to treat or prevent injury resulting from reoxygenation.
As mentioned in the Introduction section, there is a
remarkable dearth of literature dealing with the inhibitor L-NAME in lungs submitted to hypoxia/reoxygenation, making its possible effects on this organ completely
unknown. To the best of our knowledge, this is the first
study that describes the time-course effects of this nonselective NOS inhibitor in the hypoxic adult rat lung.
Alterations in the production of NOS-derived NO are
critical in the injury that occurs during hypoxia/reoxyge-
DUAL ROLES OF NO IN THE HYPOXIC LUNG
Fig. 6. (A–H): Micrographs showing nitrotyrosine immunoreactivity
in rat lung sections in endothelial cells (arrows) and bronchiolar epithelial cells (arrow heads). Experimental groups: control (C) and 0 h, 48 h,
and 5 days (5d) posthypoxia; control (C) L-NAME and 0 h L-NAME, 48
h L-NAME, and 5 days (5d) L-NAME posthypoxia. Micrographs (E) and
(G) show higher nitrotyrosine staining intensity when compared with
the control group in the untreated groups. Micrographs (F) and (H)
show less nitrotyrosine staining intensity in relation to the corresponding untreated groups.
apoptosis as on the lipid peroxidation level in the rat
lung: the percentage of apoptotic cells raised at 0 h of
reoxygenation, but decreased at 48 h and 5 days. We
have previously reported that eNOS increased in the
hypoxic rat lung at the earliest reoxygenation time (0 h),
while the inducible isoform augmented from 48 h to 5
days posthypoxia (Rus et al., 2010b). All these findings
together suggest that eNOS-derived NO may exert a
beneficial role against the lipid peroxidation and apoptosis processes, which occur during hypoxia/reoxygenation
in the lung, while iNOS-derived NO appears to be
involved in both of these harmful phenomena.
As stated above, currently there are only scarce and
controversial studies dealing with NOS inhibitors in the
hypoxic lung, and they mostly are performed in cultured
cells, not in vivo, as this work. In this sense, while some
authors showed that inhibition of NO synthesis by
L-NAME decreased greatly the production of lung
TBARS after ischemia/reperfusion (Ischiropoulos et al.,
2199
1995), others found that the same NOS inhibitor
increased the concentrations of TBARS after intermittent hypoxia in hamster cheek pouch microcirculation
(Bertuglia, 2008). Regarding apoptosis, Maejima et al.
(2003) reported that L-NAME greatly increased the apoptosis rate in neonatal rat cardiomyocytes under ischemia/reperfusion, indicating that the NO released during
these situations exerts an antiapoptotic effect. By contrast, Yang et al. (2005) found that hypoxia/reoxygenation-induced apoptosis was significantly decreased by
L-NAME in liver sinusoidal endothelial cells, and Viñas
et al. (2006) showed that the number of TUNEL-positive
cells, increased by ischemia/reperfusion, decreased with
L-NAME in rat kidney, suggesting a role for NO in the
development of apoptosis. In fact, it has been previously
reported that NO can exert both pro- and antiapoptotic
effects. In this way, NO can act as a second messenger,
activating a number of cytokines that induce apoptosis
(Saugstad, 2000), or it can inhibit aconitase in the tricarboxylic acid cycle, leading to a glycolysis inhibition (Stadler et al., 1991). On the contrary, NO can also inhibit
apoptosis by nitrosylating active sites of cysteine residues in caspases, essential enzymes for the apoptotic
process (Leist et al., 1999).
Finally, our results have also shown that our model of
hypoxia/reoxygenation significantly augmented nitrotyrosine-modified protein expression at 48 h, and 5 days posthypoxia in the untreated groups. However, the treatment
with the NOS inhibitor L-NAME significantly decreased
nitrated proteins at those same times, suggesting an
involvement of NOS-derived NO in nitrotyrosine formation
in the hypoxic lung. This decrease in nitrated protein
expression is consistent, since L-NAME reduces NOx levels, and as a result, the formation of peroxinitrite, diminishing the nitrotyrosine-modified proteins formation.
These results were corroborated by the nitrated protein
location data, found in vascular endothelial cells and bronchiolar epithelium. There are, nonetheless, controversial
results around the effects of this nonselective NOS inhibitor on nitrotyrosine levels in other organs and models. In
this sense, Ischiropoulos et al. (1995) reported that the
treatment with L-NAME counteracted the rise in nitrotyrosine levels after ischemia/reperfusion in the lung, and Serrano et al. (2006) showed that the administration of the
same NOS inhibitor prevented the increase in nitrotyrosine immunoreactivity provoked by hypobaric hypoxia in
rat cerebral cortex. On the contrary, other authors demonstrated that nitrotyrosine increased in the groups treated
with L-NAME in the acutely hypoxic brain (Litt et al.,
1999).
In summary, the results of this study demonstrate that a
treatment with the nonselective NOS inhibitor L-NAME in
hypoxic lungs exacerbates lipid peroxidation and apoptosis
just after the hypoxic stimulus, but decreases them in the
later posthypoxia times. These findings suggest that NOSderived NO exerts two different effects on lung hypoxia/
reoxygenation-induced damage depending on the reoxygenation time: NO may have a beneficial role at the earliest
posthypoxia period and a deleterious role in the later reoxygenation times. Moreover, this dual role of NO may
depend directly on the producer NOS isoform: while eNOSderived NO would exert a protective effect against the injuries which occur during hypoxia/reoxygenation in the lung,
iNOS-derived NO appears to have a detrimental role. We
suggest that the administration of L-NAME would be
2200
RUS ET AL.
beneficial for the treatment of lung hypoxia/reoxygenation
injuries after 48 h posthypoxia.
ACKNOWLEDGMENTS
The authors thank Rafael Lomas for his statistic assistance, and David Nesbitt for his editorial help with the
English version of the manuscript.
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