Regulation of diaphragmatic nitric oxide synthase expression
during hypobaric hypoxia
Danesh
Javeshghani1,
Dalia
Sakkal1,
Mastaka
Mori2, and
Sabah N. A.
Hussain1
1 Critical Care and Respiratory Divisions, Royal Victoria
Hospital and Meakins-Christie Laboratories, McGill University,
Montreal, Quebec, Canada H3A 1A1; and 2 Department of
Molecular Genetics, Kumamato University, School of Medicine, Kumamato
862, Japan
 |
ABSTRACT |
Nitric oxide (NO) is normally synthesized
inside skeletal muscle fibers by both endothelial (eNOS) and neuronal
(nNOS) nitric oxide synthases. In this study, we evaluated the
influence of hypobaric hypoxia on the expression of NOS isoforms,
argininosuccinate synthetase (AS), argininosuccinate lyase (AL), and
manganese superoxide dismutase (Mn SOD) in the ventilatory muscles.
Rats were exposed to hypobaric hypoxia (~95 mmHg) from birth for 60 days or 9-11 mo. Age-matched control groups of rats also were
examined. Sixty days of hypoxia elicited approximately two- and
ninefold increases in diaphragmatic eNOS and nNOS protein expression
(evaluated by immunoblotting), respectively, and about a 50% rise in
diaphragmatic NOS activity. In contrast, NOS activity and the
expression of these proteins declined significantly in response to 9 mo
of hypoxia. Hypoxia elicited no significant alterations in AS, AL and
Mn SOD protein expression. Moreover, the inducible NOS (iNOS) was not detected in normoxic and hypoxic diaphragmatic samples. We conclude that diaphragmatic NOS expression and activity undergo significant adaptations to hypobaric hypoxia and that iNOS does not participate in
this response.
respiratory muscles; mitochondria; oxygen radicals
 |
INTRODUCTION |
NITRIC
OXIDE (NO), a secondary messenger with diverse biological
functions, is normally synthesized in skeletal muscles by neuronal
(nNOS) and endothelial (eNOS) nitric oxide synthases. Recent studies
have confirmed that while the nNOS isoform is localized at the
sarcolemma of type II fibers and closely associates with the dystrophin
complex (9), the eNOS isoform is mainly localized in the
endothelium of skeletal muscle vasculature. The functional significance
of both nNOS and eNOS isoforms in regulating skeletal muscle function
is under investigation. However, increasing evidence suggests that many
processes inside skeletal muscle fibers such as blood flow, glucose
uptake, myoblast fusion, and excitation-contraction coupling are
influenced by endogenous NO synthesis (1, 6, 18, 30, 31,
35).
The diaphragm is a skeletal muscle that is exposed to loads with
magnitudes that depend on the mechanical characteristics of the
respiratory system and timing, which are related to breathing frequency. As with other skeletal muscles, it has been well established that both nNOS and eNOS isoforms are expressed in the diaphragm of
various species (15, 16, 19). More recent studies by our
group demonstrated that the rate of NO synthesis in skeletal muscle
fibers is a dynamic process that undergoes specific alterations in
response to changes in muscle metabolic demands. For instance, an
increase in muscle activity over a relatively short period (3 h of
resistive loading) initiates a significant reduction of muscle NO
synthesis (12). Muscle NO synthesis and eNOS and nNOS expression are also elevated during embryonic and postnatal
development (10).
Hypobaric hypoxia is associated with numerous humoral, nutritional, and
respiratory changes such as increased ventilatory muscle demands, which
result in significant biochemical and histological adaptations in the
ventilatory muscles in general and in the diaphragm in particular. For
instance, diaphragmatic myosin heavy chain isoforms change
significantly in chronically hypoxic rats as indicated by a decrease in
type I isoform expression and an increase in expression of the type II
isoform (23). Similar alterations were detected in the
quadriceps of chronic obstructive lung disease patients
(14).
Many investigators recently have addressed the influence of relatively
short periods of hypoxia on NO synthesis and have reported specific
changes in NOS expression, which depend on the NOS isoform and the type
of cell that is involved. There is no information regarding the
influence of hypobaric hypoxia on the expression of various NOS
isoforms inside the ventilatory muscles. We hypothesized that moderate
durations of hypobaric hypoxia (60 days) lead to a significant
upregulation of both nNOS and eNOS expression in the ventilatory
muscles. This hypothesis is based on the following observations. First,
hypobaric hypoxia promotes a shift toward type II fibers that are rich
in the nNOS isoform (17). Second, hypobaric hypoxia is
associated with increased ventilatory muscle activity due to
augmentation of minute ventilation, which results in increased
ventilatory muscle metabolic demands (23). Previous studies indicate that prolonged periods of increased skeletal muscle
activity leads to a substantial rise in muscle nNOS expression (28). Third, hypobaric hypoxia causes vascular remodeling
(smooth muscle and endothelial cell replication and extracellular
matrix accumulation), which is mediated by vascular cell mitogens and growth factors such as vascular endothelial cell growth factor (VEGF),
endothelin-1, and platelet-derived growth factor. These mitogens,
particularly VEGF, are known to stimulate eNOS expression in the
endothelial cells (20). On the other hand, we propose that
prolonged (over several months) hypobaric hypoxia will be associated
with downregulation of both nNOS and eNOS isoforms in the ventilatory
muscles because of various nutritional and hormonal alterations
associated with prolonged hypoxia. These alterations include a decline
in thyroid function. Recent studies indicate that NOS expression is
positively modulated by the thyroid hormone (34). The main
objective of this study, therefore, was to test whether moderate (60 days) and prolonged (9 mo) periods of hypobaric hypoxia elicit
differential effects on constitutive NOS isoform expression in the
diaphragm and whether hypobaric hypoxia induces the expression of the
inducible NOS (iNOS). Finally, we evaluated the influence of hypobaric
hypoxia on the expression of two enzymes involved in the recycling
of L-citrulline to L-arginine [argininosuccinate synthase (AS) and argininosuccinate lyase (AL)].
The study was conducted with a model that was used previously to
address changes in diaphragmatic fiber-type expression in response to
chronic hypoxia (23). Conscious rats were exposed to a
moderate level of hypobaric hypoxia for 60 days or 9 mo after birth.
With the use of immunoblotting, we examined the relative changes in
diaphragmatic eNOS, nNOS, iNOS, AS, and AL protein expression. We also
probed diaphragmatic muscle samples with an antibody selective to
manganese superoxide dismutase (Mn SOD) to evaluate the possibility
that changes in NOS isoform expression in hypoxic rats represent a
general adaptative behavior involving not only NO synthesis but also
antioxidant enzymes such as Mn SOD.
 |
MATERIALS AND METHODS |
Animal preparation.
All experiments were approved by the appropriate Animal Ethics
Committees of McGill University. Experiments were performed on two
groups of adult Sprague-Dawley rats, normoxic and chronic hypoxic.
Animals were studied either at 60 days of age (n = 8 per group) or after 9-11 mo of age (n = 6 per
group). The hypoxic group was continuously exposed to hypobaric hypoxia
from the first day after birth. Hypobaric hypoxia (barometric pressure
of ~505 mmHg, which corresponds to PO2 of
~95 mmHg) was established in chambers that were opened twice a week
for 20 min to clean and replenish food and water. The animals were
weaned (days 22-23) and were then housed in
separate cages containing one or more animals and allowed free access
to food and water. The animals were exposed to a daily 12:12-h
light-dark cycle and kept under ambient temperature and humidity. The
normoxic group was housed in similar chambers at barometric pressure of
760 mmHg (PO2 150 mmHg) and were maintained
under conditions similar to those of the hypoxic group.
Sample preparation.
The animals were anesthetized with 30 mg/kg pentobarbital sodium.
Bleeding was then initiated by severing the carotid arteries and
jugular veins. The abdomen was opened, and the costal diaphragm was
removed, blotted on absorbing paper, weighed, and stored under
80°C. About 100 mg of muscle sample were homogenized by hand in a
glass tissue grinder with ice-cold homogenization buffer containing
0.2% sodium dodecyl sulfate, 0.6 M
-mercaptoethanol, 28 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, 22 mM Tris base,
0.002% leupeptin, 250 mM phenylmethylsulfonyl fluoride, and 0.5%
trypsin inhibitor. The sample was then heated at 95°C for 12 min,
placed on ice for 30 min, and centrifuged (10,000 g), and
the supernatant was then collected. The resultant pellet was
resuspended in the homogenization buffer, and the procedure was
repeated three to four times until the protein concentration of the
resuspended pellet was <5% of the protein concentration of the total
supernatant. The procedure was used to extract as much protein as
possible from muscle samples. Supernatants from repeated centrifugation
were pooled, and protein concentration was measured with the Bradford
technique (Bio-Rad).
Immunoblotting.
Crude muscle homogenate proteins (80 µg; see above) were heated for
15 min at 90°C and then loaded on gradient (4-12%) sodium dodecyl sulfate-Tris glycine polyacrylamide gels. The proteins were
transferred electrophoretically onto polyvinylidene difluoride membranes and were blocked with 5% nonfat dry milk and subsequently incubated overnight at 4°C with primary monoclonal anti-iNOS (1:500), anti-eNOS (1:500), and anti-iNOS (1:500) antibodies (all obtained from
Transduction Laboratories, Lexington, KY). Lysates of
cytokine-activated macrophages, endothelial cells, and pituitary cells
were used as positive controls for iNOS, eNOS, and nNOS proteins,
respectively (provided by Transduction Laboratories). AS and AL
proteins (1:1,000) were detected with polyclonal antibodies that were
raised in rabbits against recombinant proteins and were used previously
to detect these proteins in the diaphragm and other rat tissues
(24). A polyclonal antibody raised against rat Mn SOD
(1:500) was used to detect diaphragmatic Mn SOD expression
(33). Specific proteins were detected with horseradish
peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies
and enhanced chemiluminescence reagents (Amersham).
Predetermined molecular weight standards (Novex) were used as markers.
The membranes were stained with silver stain and scanned to verify that
equal amounts of proteins were loaded on different lanes.
NOS activity.
Diaphragmatic NOS activity was measured with the
L-citrulline assay (10). Frozen tissues were
homogenized in 6 volumes (wt/vol) of homogenization
buffer (pH 7.4, 10 mM HEPES buffer, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml phenylmethylsulfonyl fluoride, 0.32 mM sucrose, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin A). The crude
homogenates were centrifuged at 4°C for 15 min at 10,000 rpm. The
supernatant (50 µl) was added to 10-ml prewarmed (37°C) tubes
containing 100 µl of reaction buffer of the following composition: 50 mM KH2PO4, 60 mM valine, 1.5 mM NADPH, 10 mM
flavine adenine dinucleotide, 1.2 mM MgCl2, 2 mM CaCl2, 1 mg/ml BSA, 1 µg/ml calmodulin, 10 µM
tetrahydrobiopterin, and 25 µl of 120 µM stock
L-[2,3-3H]arginine (150-200
counts · min
1 · pM
1). The
samples were incubated for 30 min at 37°C, and the reaction was
terminated by the addition of cold (4°C) stop buffer (pH 5.5, 100 mM
HEPES and 12 mM EDTA). To obtain free
L-[3H]citrulline for the determination of
enzyme activity, 2 ml of Dowex 50 W resin (8% cross-linked,
Na+ form) were added to eliminate excess
L-[2,3-3H]arginine. The supernatant was
assayed for L-[3H]citrulline by using liquid
scintillation counting. Enzyme activity is expressed in picomoles of
L-citrulline produced per minute per milligram of total
protein. NOS activity was also measured in the presence of 1 mM
NG-nitro-L-arginine methyl ester
(L-NAME; NOS inhibitor). Total NOS activity was calculated
as the difference between that measured in the absence and presence of
L-NAME.
Data analysis.
Protein band intensities were quantified by scanning blots containing
six samples per given animal group. Scanning was performed with an
imaging densitometer (model GS700, Bio-Rad, 12-bit precision and
42-µm resolution). Optical densities of the protein bands were
quantified with SigmaGel software (Jandel Scientific, San Rafael, CA).
Densities and NOS activity values were compared between normoxic and
hypoxic groups with two-way analysis of variance. P < 0.05 was considered significant.
 |
RESULTS |
Sixty days.
Chronic hypoxia for 60 days had no effect on body weight or
diaphragmatic mass (as percentage of body mass; Table
1). Hematocrit, however, increased
significantly in the hypoxic group compared with that in the normoxic
group (Table 1).
Immunoblotting of diaphragmatic proteins with anti- nNOS
antibody detected a prominent band at an apparent mass of 166 kDa (Fig.
1). At 60 days of age, hypoxia was
associated with more than ninefold induction of nNOS protein (Fig. 1).
Anti-eNOS antibody detected a single protein band at 130 kDa, which was
upregulated by an approximate twofold in response to 60 days of hypoxia
(Fig. 2). iNOS protein was not detected
in the normoxic and hypoxic groups (Fig.
3). Furthermore, 60 days of hypoxia
elicited no significant changes in AS, AL, and Mn SOD protein
expression. Figure 4 illustrates the mean
values of optical densities of eight different diaphragmatic samples in
each group of animals. Hypoxia was associated with an approximate two-
and ninefold rise in the expression of eNOS and nNOS, respectively,
whereas no significant changes were detected in AS, AL, and Mn SOD
expressions. Figure 5 illustrates the
changes in diaphragmatic NOS activity. Hypobaric hypoxia for 60 days
resulted in a significant increase in diaphragmatic NOS activity (Fig. 5).

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Fig. 1.
Changes in diaphragmatic neuronal nitric oxide synthase
(nNOS) protein expression in the normoxic and hypoxic animals at 60 days of age. Note the rise in nNOS protein expression in the hypoxic
group compared with that in the normoxic group.
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Fig. 2.
Influence of 60 days of hypoxia on diaphragmatic
endothelial NOS (eNOS) protein expression. The expression of eNOS
protein in the hypoxic group was about 2 times that observed in the
normoxic group.
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Fig. 3.
Expression of inducible NOS (iNOS) in the normoxic and hypoxic
animals. +ve, Positive control protein sample (cytokine-activated
macrophages). Note that iNOS protein was detected neither in the
hypoxic nor in the normoxic groups.
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Fig. 4.
Optical densities of various proteins detected in the
normoxic and hypoxic groups after 60 days of age. Mn SOD, manganese
superoxide dismutase; AS, argininosuccinate synthetase; AL,
argininosuccinate lyase. Values are means of 6 different samples per
group. ** P < 0.01 compared with the normoxic
group.
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Fig. 5.
Changes in diaphragmatic NOS activity in the 4 groups of
animals. * P < 0.05 and ** P < 0.01 compared with corresponding normoxic groups.
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Nine to eleven months.
Although body weight and diaphragmatic mass were not different among
the two groups, hematocrit rose significantly in the hypoxic group
compared with that in the normoxic group (Table 1). Prolonged
(9-11 mo) periods of hypoxia elicit (qualitatively and
quantitatively) different changes in diaphragmatic NOS expression than
those associated with shorter periods of hypoxia (60 days). Prolonged
hypoxia reduced nNOS protein expression, which averaged about 20% of
that observed in the normoxic group (Fig.
6). Similarly, diaphragmatic eNOS
expression was lower in the hypoxic group and averaged about 15% of
that observed in the normoxic group (Fig. 6). Expression of AS, AL, and
Mn SOD was not altered by prolonged hypoxia. Figure
7 shows the mean values of optical
densities of six different diaphragmatic samples in each group of
animals. Prolonged hypobaric hypoxia (9 mo) resulted in a significant
attenuation of diaphragmatic NOS activity compared with that in
normoxic animals (Fig. 5).

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Fig. 6.
The influence of 9 mo of hypoxia on diaphragmatic nNOS
and eNOS protein expression. Note that the levels of both proteins
declined significantly in the hypoxic group compared with those in the
normoxic group.
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Fig. 7.
Mean values of optical densities of various proteins in response to
9 mo of hypoxia. ** P < 0.01 compared with the
normoxic group.
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 |
DISCUSSION |
The main finding of this study is that chronic hypobaric hypoxia
elicits selective changes in diaphragmatic NOS expression and activity
and that those changes are related to the duration of hypoxic exposure.
Sixty days of hypobaric hypoxia in newborn rats was associated with
upregulation of eNOS and nNOS protein levels and an increase in
diaphragmatic NOS activity, whereas 9 mo of hypoxia reduced the
expression of both enzymes and attenuated NOS activity. The changes in
diaphragmatic NOS expression in response to hypoxia are limited to the
constitutive isoforms because iNOS was not induced by exposure to
hypobaric hypoxia. In addition, hypoxia had no effects on diaphragmatic
expressions of AS, AL, and Mn SOD proteins.
Regulation of eNOS and nNOS expression by hypoxia.
Studies on the influence of relatively short periods (a few hours to 3 wk) of hypoxia on constitutive NOS isoform expression revealed
conflicting results depending on the animal species, the duration of
hypoxia, and the cell and organ involved. For example, 12-48 h of
in vitro hypoxia elicited a decline in pulmonary endothelial cell eNOS
expression, whereas eNOS expression in aortic endothelial cells rose
significantly (3, 21). Similarly, fetal pulmonary eNOS
expression declines significantly in response to 48 h of in vitro
hypoxia, whereas bronchiolar epithelial NO production remains
independent of PO2 levels (25).
Contrasting results also have been reported regarding the influence of
in vivo hypoxia on lung eNOS expression. Although Xue et al.
(37) demonstrated a substantial upregulation of eNOS
protein expression in pulmonary endothelial cells and de novo induction
of NOS expression in vascular smooth muscles in response to 2-4 wk
of hypoxia in rats, whole lung eNOS protein expression after 10-12
days of hypoxia in newborn piglets was only about 40% of that observed
in normoxic animals (11). The reasons behind these
contradictory results regarding eNOS expression are not clear. It is
likely that a combination of a direct influence of hypoxia on an eNOS
promoter and/or secondary changes in shear stress and hormonal levels
may result in the tailoring of local NO production to meet altered NO
requirements during hypoxia.
Unlike the eNOS isoform, the influence of hypoxia on the expression of
the nNOS isoform has been shown consistently to be stimulative,
especially in the central and peripheral nervous system. For instance,
subacute hypoxia (12-48 h) in conscious rats results in
significant upregulation of both nNOS mRNA and protein expression in
the cerebellum (13). Similarly, Prabhakar et al.
(27) reported approximately 10- and 2-fold increases in
nNOS mRNA in nodose ganglia and cerebellums after 12 h of hypoxia in conscious rats, respectively.
No information is available yet regarding the influence of hypobaric
hypoxia on skeletal muscle NOS expression. Our results indicate for the
first time that prolonged periods of hypobaric hypoxia (60 days to 9 mo) elicit differential changes in constitutive NOS isoform expression,
which are related to the duration of hypoxia. After 60 days of hypoxia
in newborn rats, diaphragmatic eNOS and nNOS expression rose
significantly, whereas 9 mo of hypoxia elicited an opposite response.
The exact molecular mechanisms responsible for the changes in
diaphragmatic NOS expression in our study remain unclear. We speculate
that changes in protein levels of diaphragmatic eNOS and nNOS isoforms
are the results of transcriptional and/or posttranscriptional
mechanisms. One of these mechanisms is likely to be a change in NOS
mRNA stability. Indeed, hypoxia evokes significant shortening of eNOS
mRNA half-life in pulmonary endothelial cells (21). By
comparison, a decline in eNOS transcription was the only alteration
noted in response to severe hypoxia in aortic endothelial cells,
whereas eNOS mRNA stability remained unchanged (3). Our
results do not exclude changes in diaphragmatic eNOS and nNOS
transcription and/or alterations in mRNA stability of these isoforms in
response to 60 days or 9 mo of hypoxia.
Another issue that should be addressed is whether the aging process is
involved in the differential alterations of diaphragmatic NOS
expression in response to 60 days or 9 mo of hypobaric hypoxia. Previous studies revealed that age-related changes in NOS expression differ among various organs. In mouse hearts, eNOS and nNOS activities increase by 120 and 47%, respectively, between 2 and 6 mo of age (5). By comparison, no significant changes were detected
in nNOS expression in the human brain during aging (7).
With respect to skeletal muscles, Richmonds et al. (29)
compared NOS activity in various muscles of 8- and 24-mo-old rats and
reported a significant reduction in NOS activity with aging. We
measured NOS activity and eNOS and nNOS expression during early
postnatal development in rats and found a significant decline in these
parameters in adult animals compared with those in newborn animals
(10). To assess the influence of aging on differential NOS
expression in our experiment, we performed immunoblotting on five
animals in each of the normoxic groups (60 days and 9 mo old) using
various antibodies listed in MATERIALS AND METHODS.
Diaphragmatic eNOS and nNOS optical densities of normoxic 9-mo-old
diaphragms averaged about 120 ± 10 and 107 ± 5%,
respectively, of those of 60-day-old normoxic diaphragms (both values
are not statistically significant). Similarly, no significant
age-related changes in diaphragmatic AS, AL, and Mn SOD expression were
detected among the two normoxic groups. We should emphasize that
diaphragmatic NOS activities were not different among the normoxic
groups (Fig. 5). These results indicate that aging does not explain the
differences in the responses of diaphragmatic NOS expression and
activity to moderate and prolonged periods of hypobaric hypoxia.
Clearly, more studies are needed to elucidate the molecular mechanisms
behind the changes in diaphragmatic NOS isoforms in response to
hypobaric hypoxia.
An interesting observation in our study is that hypoxia-induced
changes in diaphragmatic NOS activity were relatively smaller than
those of diaphragmatic NOS protein expression. For instance, whereas
diaphragmatic NOS activity rose by about 50% in response to 60 days of
hypobaric hypoxia, diaphragmatic eNOS and nNOS expression increased by
about 2.5- and 9-fold, respectively. The reasons behind this
observation are not yet clear; however, it has been established that
NOS activity is influenced not only by the level of NOS proteins but by
the availability of cofactors such as tetrahydrobiopterin and the
presence of endogenous inhibitors such as
NG,NG-dimethyl-L-arginine,
protein inhibitor of nNOS and caveolin 3. We speculate that
the disproportionate changes in muscle NOS activity compared with NOS
protein expression in this study are due to hypoxia-induced alterations
in the levels of NOS cofactors and endogenous inhibitors. Clearly, more
studies are needed to elucidate the direction and the mechanisms
through which hypoxia may influence the availability and the expression
of NOS cofactors and endogenous inhibitors.
It should be emphasized that one should be cautious in extrapolating
the observed changes in diaphragmatic constitutive NOS expression in
response to hypobaric hypoxia to other skeletal muscle because only the
diaphragm was examined in the current study. We speculate, however,
that similar changes in NOS expression are likely to develop in the
intercostal muscles in response to hypobaric hypoxia. Our previous
studies in spontaneously breathing animals and in animals exposed to
inspiratory resistive loading or sepsis indicate that NOS expression
and activity in the intercostal muscles were similar to those of the
diaphragm (15, 16).
Induction of iNOS, AS, and AL by hypoxia.
Under normal conditions, very weak iNOS expression is detectable in the
ventilatory or limb muscles of various species. However, exposure to
bacterial lipopolysaccharides induces significant iNOS expression in
skeletal muscle fibers of various muscles (8). Induction
of iNOS is usually accompanied by increased requirements for
L-arginine, which is supplied through active transport from the extracellular space, protein degradation, and recycling of L-citrulline to L-arginine. This recycling
occurs via a two-step enzymatic process that requires the activities of
AS and AL enzymes. The expression of these enzymes has been widely
reported to be induced along with iNOS in response to proinflammatory
cytokines or lipopolysaccharides (24). Little is known
about the influence of hypoxia on iNOS, AS, and AL expression. In
murine cell lines, iNOS is induced by hypoxia only when these cells
were costimulated with interferon-
(22). In the
respiratory system, Palmer and colleagues (26) have
reported recently that 3 wk of hypoxia in conscious rats is associated
with the induction of iNOS mRNA in bronchial epithelial, pulmonary
endothelial, and smooth muscle cells (26). Induction of
iNOS by hypoxia appears to vary between different tissues. We reported
recently that no significant iNOS induction occurs in the brain in
response to 12-48 h of hypoxia in rats (13).
Similarly, few studies have addressed the influence of hypoxia on AS
and AL expression. Su and Block (32) reported that 24 h of in vitro hypoxia elicit a significant downregulation of both AS
activity and expression in cultured pulmonary endothelial cells
(32). These results contrast with our recent findings indicating no significant alterations in brain AS and AL expression in
response to 12-48 h of in vivo hypoxia in rats (13).
Our current results indicate that prolonged periods of hypobaric
hypoxia had no influence on iNOS induction in the diaphragm. These
results indicate that despite the presence of a putative hypoxia-induced element in iNOS promoter, induction of the iNOS gene in
response to in vivo hypoxia is regulated at the local level. The lack
of induction of diaphragmatic AS and AL in the present study also
suggests the link between iNOS induction and recycling of
L-citrulline to L-arginine is particularly
strong during in vivo hypoxia.
Implications.
The changes in the expression of nNOS and eNOS isoforms described in
this study are likely to have important impacts on the following
processes in skeletal muscle fibers. 1) Glucose transport. Skeletal muscle fibers, particularly neonatal or immature fibers, rely
to a greater extent on glycolysis and glucose uptake for ATP production
during hypoxia rather than during normoxia. To achieve this goal, many
steps involved in muscle glucose uptake are augmented, including the
expression of glucose transporters GLUT-1 and GLUT-4 (36).
Recent studies have confirmed that NO has an important role in muscle
glucose uptake. Indeed, basal and insulin- and contraction-stimulated
glucose uptakes are enhanced by endogenous NO synthesis (4,
18). On the basis of the aforementioned studies, one can
conclude that elevated nNOS and eNOS expression during 60 days of
hypobaric hypoxia represent an adaptative response by neonatal muscles
to cope with increased demands for glucose uptake. In addition,
reduction of NOS activity as a result of downregulation of both eNOS
and nNOS in 9-mo-old hypoxic rats is likely to have adverse effects on
glucose uptake of muscle fibers. 2) Muscle fiber maturation.
Recent studies from our laboratories and others (6, 10)
have illustrated that NO promotes myoblast fusion in skeletal muscles
during embryonic and early postnatal development. Thus increased muscle
NO synthesis during moderate periods of hypobaric hypoxia is likely to
promote myoblast fusion and maturation of neonatal muscle fibers.
3) Reactive oxygen species (ROS). Although excessive NO
release is damaging to tissues as a result of the formation of the free
radical peroxynitrite, most investigators agree that basal NO
production in skeletal muscles plays an important role as a ROS
scavenger. Recent studies indicate that NO enhances
excitation-contraction coupling by protecting sarcoplasmic reticulum
ryanodine receptors from ROS-mediated oxidative modifications,
resulting eventually in activation of these receptors (1,
31). We speculate that a reduction in muscle NO production in
response to hypobaric hypoxia will have adverse effects on muscle
antioxidant capacity and excitation-contraction particularly in
9-mo-old rats because of the already depressed antioxidant enzyme
activities in these rats compared with those in younger animals
(2). 4) Muscle blood flow. The involvement of
NO release from endothelial cells in the regulation of muscle blood
flow is very well documented. We propose that the increase in eNOS expression in response to moderate periods of hypobaric hypoxia will
promote vascular dilation and increased blood flow supply to the
developing muscle fibers.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. J. Mortola and L. Naso, Department of
Physiology, McGill University, for assistance in performing the animal
experiments and tissue collection.
 |
FOOTNOTES |
This study was funded by the Medical Research Council of Canada. S. Hussain is a scholar of the Fonds de la Recherche en Sante du Quebec.
Address for reprint requests and other correspondence: S. Hussain, Rm. L3.05, 687 Pine Ave. West, Montreal, Quebec, Canada H3A
1A1 (E-mail: sabah.hussain{at}muhc.mcgill.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. §1734 solely to indicate this fact.
Received 11 January 2000; accepted in final form 6 April 2000.
 |
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