Upregulation of lung soluble guanylate cyclase during chronic
hypoxia is prevented by deletion of eNOS
Dechun
Li1,
Victor E.
Laubach2, and
Roger A.
Johns1
1 Department of Anesthesiology and Critical Care Medicine,
Johns Hopkins University School of Medicine, Baltimore, Maryland
21287; and 2 Department of Surgery, University of Virginia
Health System, Charlottesville, Virginia 22908
 |
ABSTRACT |
Hypoxia upregulates
endothelial (e) nitric oxide synthase (NOS), but how eNOS affects
soluble guanylate cyclase (sGC) protein expression in hypoxia-induced
pulmonary hypertension is unknown. Wild-type (WT), eNOS-deficient
[eNOS(
/
)], and inducible NOS (iNOS)-deficient [iNOS(
/
)]
mice were used to investigate the effects of lack of NO from different
NOS isoforms on sGC activity and protein expression and its
relationship to the muscularization of the pulmonary vasculature. After
6 days of hypoxic exposure (10% O2), the ratios of the
right ventricle to left ventricle + septum weight (RV/LV+S) and
right ventricle weight to body weight, the lung sGC activity, and
vascular muscularization were determined, and protein analysis for
eNOS, iNOS, and sGC was performed. Results demonstrated that there were
significant increases of RV/LV+S in all animals treated with hypoxia.
In hypoxic WT and iNOS(
/
) mice, eNOS and sGC
1- and
1-protein increased twofold; cGMP levels and the number
of muscularized vessels also increased compared with hypoxic
eNOS(
/
) mice. There was a twofold increase of iNOS protein in WT
and eNOS(
/
) mice, and the basal iNOS protein concentration was
higher in eNOS(
/
) mice than in WT mice. In contrast, the eNOS(
/
) mouse lung showed no eNOS protein expression, lower cGMP
concentrations, and no change of sGC protein levels after hypoxic
exposure compared with its normoxic controls (P > 0.34). These results suggest that eNOS, but not iNOS, is a major
regulator of sGC activity and protein expression in the pulmonary vasculature.
nitric oxide; vascular remodeling; pulmonary hypertension; endothelial nitric oxide synthase
 |
INTRODUCTION |
STUDIES HAVE
DEMONSTRATED that the nitric oxide (NO)-cGMP signaling pathway
participates in the vascular remodeling process in hypoxia-induced
pulmonary hypertension (1, 9, 10, 17, 24, 27, 30-32,
34). NO regulates the vascular tone and resistance in the
pulmonary circulation and plays an important role in the regulation of
vascular smooth muscle cell proliferation, migration, and
differentiation in the developing process of pulmonary hypertension both in animals and in humans (11, 18, 20, 35). Congenital disruption of endothelial nitric oxide synthase (eNOS) resulted in mild
and persistent systemic and pulmonary hypertension (10, 14, 15,
30, 31). Moreover, in chronic hypoxia-treated rats, there is an
upregulation of eNOS and soluble guanylate cyclase (sGC) gene
expression, accompanied by increased sGC activity and elevated cGMP
levels in the lung (17, 19, 34). However, there is
controversy about the role of NO in the development of vascular
remodeling in hypoxia-induced pulmonary hypertension. Steudel et al.
(30, 31) and Fagan et al. (10) have
demonstrated that there are increased pulmonary pressure and resistance
and more remodeling in the eNOS-deficient [eNOS(
/
)] mouse
compared with the wild-type (WT) mouse after 6 wk of hypoxia exposure, presumably secondary to the reduced production of NO in the
vasculature. In contrast, a recent study from our laboratory
demonstrated that there is a reduced bromodeoxyuridine labeling index
and less muscularization in the pulmonary vasculature in an eNOS(
/
)
mouse treated with 4 and 6 days of hypoxia compared with WT animals
(25). In addition, the vasodilating effects of NO are
hindered in chronic hypoxia-treated animals (6, 28).
Inhibition of sGC in isolated rat lungs with chronic hypoxia-induced
pulmonary hypertension augmented the pulmonary pressure and resistance
(13). These results support the concept that the NO-cGMP
signaling pathway modulates pulmonary artery pressure and resistance
and participates in the process of vascular remodeling during the
development of chronic hypoxia-induced pulmonary hypertension. However,
how eNOS expression and how NO derived from different NOS isoforms
affects sGC protein expression and modulates its activity in chronic
hypoxia-induced pulmonary hypertension are still unknown.
In this study, the protein expression of eNOS, inducible nitric oxide
synthase (iNOS), and sGC and enzyme activity of sGC were investigated
in chronic hypoxia-treated WT, eNOS(
/
), and iNOS-deficient
[iNOS(
/
)] mice, and muscularization of the vasculature (<80 µm
in diameter) in these mouse lungs was compared. The results demonstrated that chronic hypoxia induces a parallel upregulation of
eNOS and sGC protein expression and increased cGMP levels in WT and
iNOS(
/
) mouse lungs but not in eNOS(
/
) mice treated with
chronic hypoxia. There is a low level of sGC protein expression and
lower cGMP concentrations in eNOS(
/
) mouse lungs compared with WT
and iNOS(
/
) mice.
 |
MATERIALS AND METHODS |
Animal exposures.
WT(+/+) C57BL/6J mice were obtained from Jackson Laboratories (Bar
Harbor, ME). eNOS(
/
) and iNOS(
/
) mice (10-16 wk old) were
generated as previously described (16, 29). All
gene-deficient animals used in this study were backcrossed at least
seven generations to the parental C57BL/6J strain to minimize the
genetic differences between the WT mice and the eNOS(
/
) and
iNOS(
/
) mice. The animals were exposed to either normoxia (room
air) or hypoxia (10% O2, normobaric), as previously
described (17, 19, 25), for 6 days (n = 6 for each normoxic group and n = 8 for each hypoxic group). Animals were maintained at 22-24°C in a room with a
12:12-h light-dark cycle. All animals were fed standard mice chow and water ad libitum and were treated humanely in accordance with institutional and federal guidelines.
Tissue collection and weight measurement of right ventricle and
left ventricle plus septum.
Preliminary hypoxia studies demonstrated that there was an increased
eNOS and sGC expression starting at 2 days of hypoxic exposure that
reached a peak at 6 days of hypoxia treatment. For the observation of
maximal sGC protein expression, we chose to use a 6-day exposure to
hypoxia in this study. After 6 days of exposure to either normoxia or
hypoxia, the mice were anesthetized with ketamine (100 mg/kg ip), and
total body weight was measured with a Mettler AJ100 balance (Mettler
Instruments, Hightstown, NJ). Next, the chest cavity was opened, and
the lungs were perfused with 2-4 ml of heparinized saline through
the pulmonary artery until the lungs appeared white. The lungs and
hearts were removed, and the left lungs were slowly inflated through
the trachea with 4% paraformaldehyde in PBS (GIBCO BRL, Grand Island,
NY) until the edge of the pleura became sharp and were fixed for 4 h before processing for paraffin-embedded sections as previously
described (19). The right lung lobe was snap-frozen in
liquid nitrogen and stored at
70°C for cGMP measurement and Western
blot analysis. The hearts from normoxia- and hypoxia-treated groups
were taken, and the right ventricle and left ventricle plus septum were
dissected and weighed. The ratio of the right ventricle to the left
ventricle + septum weight (RV/LV+S) and the ratio of the right
ventricle weight to body weight [RVW (mg)/BW (g)] were calculated and
are used as an indicator of increased pulmonary pressure and resistance.
Measurement of sGC activity in lung tissue
homogenates.
sGC activity in the animals treated with 6 days of normoxia or hypoxia
was measured as described by Mittal (21). Right lung tissue (20 mg/animal) was homogenized in buffer containing 50 mM
Tris · HCl (pH 7.6), 1 mM EDTA, 1 mM dithiothreitol, and 2 mM
phenylmethylsulfonyl fluoride. Extracts were centrifuged at 15,000 g for 30 min at 4°C. Protein concentrations were
determined using the Bio-Rad protein assay as described by Bradford
(3) and others (19). Supernatants containing
50 µg of protein were incubated for 10 min at 37°C in a reaction
mixture containing 50 mM Tris · HCl (pH 7.5), 4 mM
MgCl2, 0.5 mM IBMX, 7.5 mM creatine phosphate, 0.2 mg/ml
creatine phosphokinase, and 1 mM GTP. The reaction was terminated by
the addition of HCl to a final concentration of 0.1 N. cGMP in the
reaction mixture was measured using an RIA described previously
(19). sGC enzyme activity is expressed as picomoles of
cGMP produced per minute per milligram of lung protein.
Immunohistochemical staining for muscularization of pulmonary
vessels.
After deparaffinization, the sections (5 µm) were stained with
antibodies specific for von Willebrand factor (VWF, 1:200 dilution, for
the staining of endothelium; DAKO, Carpinteria, CA) and smooth muscle
(SM)
-actin conjugated with alkaline phosphatase (1:100 dilution,
for the staining of smooth muscle cells; Sigma, St. Louis, MO) to
differentiate these cell types in the vasculature, as previously
described (34). In brief, tissue sections were incubated
overnight at 4°C with a rabbit polyclonal antibody for VWF. The
sections were subsequently incubated with a goat anti-rabbit antibody
labeled with horseradish peroxidase (HRP; Bio-Rad, Hercules, CA) and
anti-SM
-actin conjugated with alkaline phosphatase antibodies. VWF
staining was visualized using the 3'-diaminobenzidine substrate kit
(Vector, Burlingame, CA), which produces a brown/black color, whereas
SM
-actin staining was accomplished using the new fuchsin (DAKO)
substrate to produce a red-colored precipitate in the smooth muscle
cells. All of the substrate incubation was controlled at 6 min, and the
sections were then counterstained briefly with methyl green or
hematoxylin before being mounted with Permount (Fisher, Pittsburgh, PA).
Vessel morphometry.
All analysis of vessel morphometry was performed using a blind code.
Because the pulmonary resistance mainly comes from the small resistance
vessels, the degree of muscularization of small (
80 µm in diameter)
vessels was determined in a minimum of 80-160 small vessels/animal
lung. Vessels were classified as nonmuscular (NM), partly muscular
(PM), or fully muscular (FM) by SM
-actin staining. Nonmuscularized
vessels were those that only showed positive staining for VWF (i.e.,
only stained the endothelial cells and no staining for SM
-actin was
apparent in the vessels). Partially muscularized vessels were defined
as those exhibiting at least one smooth muscle cell but no continuous
media. Fully muscularized vessels displayed a complete continuous
smooth muscle media that formed a circular ring. Total muscularized
vessels were obtained by the addition of the numbers of partly or fully muscularized vessels classified by the criteria described above. The
NM, PM, and FM vessel numbers were expressed as a percentage of the
total vessel numbers counted from the section. Computer-assisted image
analysis hardware and Image-Pro software (Media Cybernetics, Silver
Spring, MD) were used to assist the morphometric measurements of the
vessel sizes.
Western blot analysis for eNOS and sGC
protein.
The details of Western blotting analysis have been published previously
(19). Briefly, the right lobe from each mouse lung was
homogenized in homogenization buffer and centrifuged, and the protein
content was analyzed by the method of Bradford (3). Protein (100 µg) from the mouse lung was electrophoresed in a 7.5%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane
(Bio-Rad). The blots were incubated with a monoclonal anti-eNOS or
anti-iNOS antibody (1:1,000 dilution for each antibody; Transduction
Laboratories, Lexington, KY) or polyclonal sGC primary antibody (1:500
dilution; Cayman Chemicals, Ann Arbor, MI) followed by a goat
anti-mouse or anti-rabbit HRP-labeled secondary antibody (Bio-Rad).
eNOS, iNOS, and sGC protein signals were detected using an enhanced
chemiluminescence detection (Amersham) reagent. The relative amounts of
eNOS, iNOS, and sGC
1- and
1-subunits
were quantitated with a densitometer using ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). All Western blots were repeated
three times for each protein detection.
Statistical analysis.
All data are expressed as means ± SE and were analyzed for
statistical significance using ANOVA with multiple comparison testing. P
0.05 was considered as a significant difference.
 |
RESULTS |
RV/LV+S and RVW/BW.
The ratios RV/LV+S and RVW (mg)/BW (g) were measured from the groups of
WT, eNOS(
/
), and iNOS(
/
) mice treated with 6 days of normoxia
or hypoxia. In the hypoxia-treated mice, there was a significant
increase in RV/LV+S in WT, eNOS(
/
), and iNOS(
/
) mice compared
with their normoxic counterparts (P < 0.05). Both hypoxic eNOS(
/
) and iNOS(
/
) mice showed the smallest increase in the ratio of RV/LV+S compared with WT mice (P < 0.05, Fig. 1). WT animals showed the
largest increase in the RV/LV+S compared with eNOS(
/
) and
iNOS(
/
) mice (P < 0.05). In contrast, RVW (mg)/BW
(g) was increased more significantly in both WT and iNOS(
/
) mice
than in eNOS(
/
) mice. Comparison of RVW/BW showed that there was no
difference between WT and iNOS(
/
) mice (P = 0.78). In contrast, there was a lower right ventricle weight in eNOS(
/
) mice exposed to hypoxia compared with WT and iNOS(
/
) mice
[P < 0.005 compared with WT and iNOS(
/
) mice
treated with hypoxia, Fig. 2].

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Fig. 1.
Ratio of right ventricle to left ventricle + septum
weight (RV/LV+S) in wild-type (WT), inducible nitric oxide synthase
(iNOS)-deficient [iNOS( / )], and endothelial nitric oxide synthase
(eNOS)-deficient [eNOS( / )] mice treated with 6 days of normoxia
(open bars, n = 6 for each group) and hypoxia (solid
bars, n = 8 for each group). There was a significant
increase in right ventricle weight in all hypoxia-treated animals.
#P < 0.05 compared with all normoxic,
iNOS( / ), and eNOS( / ) hypoxic groups. *P < 0.05 compared with normoxic groups.
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Fig. 2.
Ratio of right ventricle (RV) weight (mg) to body weight
(g) in WT, iNOS( / ), and eNOS( / ) mice. There was a lesser degree
of increase in RV weight in eNOS( / ) mice treated with hypoxia
compared with WT and iNOS( / ) mice. *P < 0.05 compared with eNOS( / ) normoxic group. **P < 0.005 compared with all normoxic and eNOS( / ) hypoxic groups.
|
|
Morphological changes of vasculature after chronic hypoxia
exposure.
Staining with VWF/SM
-actin revealed that there were increased
numbers of muscularized vessels in WT and iNOS(
/
) mouse lungs
compared with normoxic groups (Fig. 3).
Morphometric analysis of the pulmonary small vessels (<80 µm in
diameter) demonstrated that there was a significant increase for both
FM and PM vessel numbers in WT and iNOS(
/
) mice treated with
hypoxia compared with normoxic controls. However, in eNOS(
/
) mice,
there was a smaller degree of increase in muscularized vessels (FM + PM) in eNOS(
/
) mice treated with 6 days of hypoxia compared with WT and iNOS(
/
) mice treated with hypoxia (Fig.
4).

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Fig. 3.
Immunohistochemical staining of smooth muscle -actin in WT
(A and B), iNOS( / ) (C and
D), and eNOS( / ) mice (E and F).
There was increased smooth muscle in hypoxia-treated animals
(B, D, and F) compared with normoxic
controls (A, C, and E). Original
magnification, ×250.
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Fig. 4.
Muscularization of pulmonary vessels (<80 µm in
diameter, fully muscular + partly muscular) in WT, iNOS( / ),
and eNOS( / ) mouse lungs after exposure to 6 days of hypoxia. There
was a smaller increase in muscularized vessels in eNOS( / ) mice
compared with WT and iNOS( / ) animals. *P < 0.05 compared with normoxic groups. #P = 0.56 compared with all normoxic groups.
|
|
sGC activity in the mouse lung.
Comparison of the sGC activity in the mouse lung homogenates revealed
that the cGMP concentrations were increased significantly (~2- to
3-fold higher, P < 0.05) in WT and iNOS(
/
) animals
treated with hypoxia compared with their normoxic counterparts. In
contrast, in eNOS(
/
) animals, the cGMP concentrations were lower in
both normoxic and hypoxic groups. In addition, the normoxic eNOS(
/
) animals showed lower cGMP concentrations compared with WT and iNOS(
/
) mice (P < 0.05; Fig.
5).

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Fig. 5.
cGMP levels in mouse lungs treated with normoxia or
hypoxia. There was a significant increase of cGMP levels in WT and
iNOS( / ) mouse lungs treated with hypoxia compared with
normoxia-treated animals. There were low cGMP concentrations in
eNOS( / ) mice treated with normoxia or hypoxia. *P < 0.05 compared with eNOS( / ) normoxic group.
#P < 0.02 compared with all normoxic and
eNOS( / ) hypoxic groups.
|
|
Western blot analysis for eNOS, iNOS,
and sGC protein.
Lung homogenates obtained from normoxia- and hypoxia-treated mouse
lungs demonstrated that there was an increase of eNOS protein at 6 days
in hypoxia-treated WT and iNOS(
/
) mice. There was no eNOS protein
detected in either normoxia- or hypoxia-treated eNOS(
/
) mice
(Fig. 6). There was no iNOS protein found
in the iNOS(
/
) mice, and iNOS protein was increased in both WT and eNOS(
/
) mice treated with hypoxia. Interestingly, the basal iNOS
protein levels were higher in eNOS(
/
) mice than in WT mice (Fig.
7). Moreover, there was a twofold
increase of sGC
1- and
1-subunit protein
that parallels the increase of eNOS protein in WT and iNOS(
/
) mice
after exposure to 6 days of hypoxia (P < 0.05, 1-way
ANOVA). However, in hypoxic eNOS(
/
) mice, the sGC protein showed no
change compared with normoxic controls (Fig. 8).

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Fig. 6.
Representative Western blot analysis of eNOS protein in WT,
iNOS( / ), and eNOS( / ) mouse lungs exposed to normoxia (N, N1,
N2, and N3) and hypoxia (H, H1, H2, and H3). eNOS protein was increased
in WT and iNOS( / ) mice treated with hypoxia. There was no eNOS
expression in eNOS( / ) mice. *P < 0.05 compared
with normoxic controls.
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Fig. 7.
Western blot analyses of iNOS protein in WT, iNOS( / ), and
eNOS( / ) mouse lung exposed to normoxia (N1, N2, and N3) and
hypoxia (H1, H2, and H3). iNOS protein was increased in WT and
eNOS( / ) mice treated with hypoxia. There was no iNOS expression in
iNOS( / ) mice. *P < 0.05 compared with normoxic
controls. #P < 0.05 compared with WT
normoxic group.
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Fig. 8.
Western blot analyses of soluble guanylate cyclase (sGC) protein in
WT, iNOS( / ), and eNOS( / ) mouse lung exposed to normoxia (N1,
N2, and N3) and hypoxia (H1, H2, and H3) for 6 days. Both sGC
1- and 1-subunits were increased in WT
and iNOS( / ) animals treated with hypoxia. There was no increase of
sGC protein in eNOS( / ) mouse lungs.
|
|
 |
DISCUSSION |
Previous studies have demonstrated upregulation of eNOS, iNOS, and
sGC protein and mRNA in the lungs from chronic hypoxia-treated rats and
mice (19, 24, 34, 36). Congenital disruption of eNOS or
inhibition of NOS resulted in hypertension in both the systemic and
pulmonary circulation, indicating the general vasodilatory role of eNOS
and its dependence on the NO levels, the activity of sGC, and integrity
of the NO-cGMP signaling pathway. However, the underlying molecular and
cellular mechanisms of hypoxia-induced pulmonary vascular remodeling
are still unknown. The use of eNOS(
/
), iNOS(
/
), and WT mice
enabled us to explore the sGC protein expression under conditions of
altered NO production from different sources. The most interesting
finding in this study is that congenital disruption of eNOS was
associated with unchanged sGC protein production and low sGC activity
in the lung in normoxic conditions and during the development of
hypoxia-induced pulmonary hypertension. In WT and iNOS(
/
) mice
treated with chronic hypoxia, the protein levels for both sGC
1- and
1-subunits and cGMP levels are
increased in parallel with the increase of eNOS protein. However, in
iNOS(
/
) mice, the basal protein levels of sGC
1- and
1-subunits are higher than in WT mice, and the sGC
activity as indicated by cGMP concentrations is the same as in WT mice.
This may indicate that the sGC in iNOS(
/
) mouse lung is in a low
activity state. In contrast, in eNOS(
/
) mice, the expression of sGC
protein and the sGC activity, as indicated in cGMP levels, were both
unchanged in chronic hypoxia-treated animals. Interestingly, there was
a lesser degree of increase in vascular muscularization that correlated with the smaller increase of RVW/BW in eNOS(
/
) mice treated with
hypoxia compared with WT and iNOS(
/
) animals. These results suggest
that eNOS or NO produced by eNOS is the main regulator of sGC activity
and protein expression. Furthermore, without NO produced by eNOS, the
sGC activity in the pulmonary vasculature was lower. In contrast, iNOS,
even though its protein expression is increased in eNOS(
/
) mice
during chronic hypoxic exposure, plays little or no role in the
regulation of sGC protein expression and its activity in
hypoxia-induced pulmonary hypertension. It is worthy to note that the
spatial relationship of endogenous NO derived from eNOS in endothelial
cells may be more efficient to stimulate sGC activity and regulate sGC
expression in smooth muscle cells in the vasculature than NO derived
from iNOS in other cell types in hypoxic conditions. This is consistent
with the work from Fagan and coworkers (11) who
demonstrated that eNOS is the major modulator of the pulmonary vascular
tone after chronic hypoxia and that iNOS only played a minor role in
the regulation of pulmonary vasoactivity under these conditions.
The role of sGC in the regulation of pulmonary vasoactivity is
controversial. Steudel et al. (30) reported that the
pulmonary circulation of eNOS(
/
) mice had impaired vasodilation to
the NO donor sodium nitroprusside and to inhaled NO in vivo but was intact in vitro when rings were preconstricted with
5-hydroxytryptamine. They concluded that there might be a defect in
smooth muscle sGC activity in the pulmonary resistance bed of
eNOS(
/
) mice or that NO had little effect on the resting pulmonary
tone. Our current results provided experimental evidence for their
speculations. In contrast, Fagan and coworkers (11)
reported that bradykinin did not reduce pulmonary perfusion pressure in
eNOS(
/
) mice and that the NO donor NONOate has the same
vasodilatory effects on WT, iNOS(
/
), and eNOS(
/
) mice in the
lung. They concluded that sGC function and other NO- and
cGMP-stimulated pathways are preserved in the pulmonary resistance
arteries of eNOS(
/
) mice. In addition, they found that the response
to inhaled NO in eNOS(
/
) mice remains intact, consistent with
preserved downstream signal transduction in vivo (10). In
the present study, our data demonstrated that there are no sGC protein
quantity differences between normoxic WT and normoxic eNOS(
/
) mice,
indicating that the constitutively expressed sGC protein is regulated
by unknown factors rather than NO and downstream components of the
NO-cGMP signaling pathway are preserved. However, without eNOS-derived
NO, the constitutively expressed sGC in the smooth muscle was in a low
activity condition and failed to upregulate in hypoxic conditions. In
either case, the smooth muscle could not counterbalance the increased
vasoconstrictive activity inflicted by hypoxia exposure. A recent study
reported by Brandes and coworkers (4) demonstrated that
there is increased nitrovasodilator sensitivity in eNOS(
/
) mice.
They also found that basal cGMP levels in aortic rings were
significantly lower (50 times) in eNOS(
/
) mice than in WT mice.
Sodium nitroprusside induced a significant cGMP accumulation in
eNOS(
/
) mice compared with WT mice. In addition, they found that
the aortic expression of the sGC
1- and
1-subunits in WT and eNOS(
/
) was identical as
determined by Western blot analysis (4). They concluded that chronic deficiency of NO in eNOS(
/
) mice restores the NO sensitivity of sGC and enhances vascular smooth muscle relaxation in
response to nitrovasodilator agents but did not change its expression.
Their results may explain the observations of Fagan et al.
(11) and are in agreement with our results in the normoxic eNOS(
/
) mouse lung.
The exact mechanism of how sGC gene expression is affected by NO is not
clear. There is evidence from studies in cultured smooth muscle cells
that sGC expression is downregulated by NO donors (12,
23). These reports have been used to at least partially explain
NO tolerance and the protective role of autoregulation of cGMP
production, particularly in septic shock conditions. In contrast, Black
and coworkers (2) demonstrated that there was a
coordinated regulation of genes of the NO and endothelin pathways during the development of pulmonary hypertension in fetal lambs (22). They found that ligation of the ductus arteriosus in
utero in lambs was associated with decreased lung expression of eNOS mRNA and protein. There was also decreased expression of sGC
1- and
1-subunit protein and increased
expression of cGMP-specific phosphodiesterase V mRNA. These reports
supported our conclusion that eNOS-produced NO may be the main
regulator or stimulator for sGC protein expression in hypoxia-induced
pulmonary hypertension, and the lack of NO-stimulated sGC production in
the pulmonary vasculature may be one of the mechanistic reasons for the
increased pulmonary pressure in eNOS(
/
) mice as already
demonstrated in eNOS(
/
) mouse aorta (4). The NO-cGMP
signaling pathway may have an autoregulatory mechanism to control
its downstream component gene expression and enzyme activity to
maintain proper vascular tone and resistance by means of feedback.
Various studies have provided conflicting data regarding the potential
role of NO in pulmonary vascular remodeling induced by hypoxia. It has
been demonstrated that hypoxia can upregulate many genes, including
growth factors and vasoconstrictors. The increased expression of growth
factor and vasoconstrictor may serve as mitogens for the pulmonary
arterial smooth muscle and facilitate vascular remodeling (5,
33). In a rat model of chronic hypoxia, upregulation of eNOS in
the small vessels of the lung precedes and progresses with the time
course of muscularization of these vessels (34, 36). In
addition, the sGC expression and enzyme activity showed a remarkable
increase in chronic hypoxia-induced pulmonary hypertension in rats.
Immunohistochemistry and in situ hybridization demonstrated that the
increased sGC protein and mRNA were mainly from newly muscularized
pulmonary arterioles that are responsible for the increased pulmonary
vascular pressure and resistance (19). In this study, we
found that, in eNOS(
/
) mice, there was no significant increase in
muscularization after 6 days of hypoxia exposure compared with that
seen in WT and iNOS(
/
) mice. Furthermore, there was a reduced RVW
(mg)/BW (g) in eNOS(
/
) mice. The reduction of RV/LV+S in
eNOS(
/
) mice might be a result of hypertrophy of the left ventricle
because of mild systemic hypertension, and this ratio may not be a
reliable parameter for the assessment of pulmonary hypertension in
eNOS(
/
) mice as in WT animals. However, a lesser degree of increase
in the RVW (mg)/BW (g) might reflect the real status of pulmonary
vascular resistance. The decreased RVW (mg)/BW (g) in eNOS(
/
) mice
could be directly related to the lesser degree of muscularization in the pulmonary vasculature. The exact role of how NO affects ventricular hypertrophy is controversial. However, a study reported by
Rouet-Benzineb et al. (26) demonstrated that 4 wk of
administration of N-nitro-L-arginine methyl
ester, a nonspecific NOS inhibitor, to hypoxia-exposed rats attenuated
pulmonary arterial pressure and RVW/BW (vs. hypoxia-exposed group).
Moreover, a recent study by de Oliveira and coworkers (7)
showed that rats with prolonged treatment with a low dose of NOS
inhibitor (7.5 mg · kg
1 · day
1 for
4-6 mo) developed cardiomyocyte hypotrophy rather than
hypertrophy. Therefore, it appears that the process of myocyte
hypertrophy depends to some extent on NO formation within the heart
during pressure overload. They proposed the following two possible
explanations: the first was based on a systemic deficiency of NO,
leading to a decrease in blood supply to the heart muscle, and the
other was based on a local deficiency of NO, causing metabolic changes in the cardiomyocyte itself. Further studies are needed for the elucidation of the role of NO in ventricular hypertrophy. Several reports have found that there were no structurally significant differences in the vessel morphology in WT and eNOS(
/
) mouse lung
in normoxic conditions (10, 30, 31). The increased pulmonary resistance and pressure in eNOS(
/
) mice might be
functional and may be a result of the reduction of NO derived from eNOS
and low activity of sGC.
In summary, our studies demonstrate that eNOS(
/
) mice treated with
hypoxia have low levels of cGMP and sGC protein compared with WT and
iNOS(
/
) mice and less remodeling in the pulmonary vasculature. We
conclude that eNOS-produced NO is the main activator or stimulator of
sGC in the pulmonary vasculature. The lower levels of sGC activity in
the eNOS(
/
) mouse lung may be one of the mechanistic reasons
accounting for the increased pulmonary resistance and pressure.
Selectively activating sGC activity and increased cGMP production may
be taken as a new strategy for the treatment of pulmonary hypertension
in the future.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant R01-HL-39706 to R. A. Johns.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. A. Johns, Dept. of Anesthesiology and Critical Care Medicine, Blalock 1415, Johns Hopkins Univ. School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287 (E-mail: rajohns{at}jhmi.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.
Received 1 August 2000; accepted in final form 3 April 2001.
 |
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