Vascular Physiology Group, Department of Cell Biology and Physiology, Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131-5218
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Past studies have
demonstrated that 17-estradiol (E2
) increases
endothelial nitric oxide (NO) synthase (eNOS) activity in uterine,
heart, and skeletal muscle and in cultured human endothelial cells.
However, little is known about E2
regulation of NO
synthesis in the pulmonary vasculature. The present study evaluated
E2
regulation of eNOS function in pulmonary arteries and
thoracic aortas. We hypothesized that E2
upregulates
vascular NO release by increasing eNOS expression. To test this,
NO-dependent vasodilation was assessed in isolated perfused lungs and
aortic rings from ovariectomized Sprague-Dawley rats treated for 1 wk
with 20 µg/24 h of E2
or vehicle. Expression of eNOS
was evaluated by Western blot and immunohistochemistry. Also, a RNase
protection assay determined eNOS mRNA levels in lung and aortic
homogenates from control and treated rats. Vasodilation to ionomycin in
lungs from the E2
-treated group was enhanced compared
with that in control animals. Endothelium-intact aortic rings from
E2
-treated animals also demonstrated augmented
endothelium-dependent dilation. Both responses were blocked with NOS
inhibition. Immunostaining for eNOS was greater in pulmonary arteries
and aortas from E2
-treated compared with control rats.
However, mRNA levels did not differ between groups. Thus we conclude
that in vivo E2
treatment augments endothelium-dependent
dilation in aorta and lung, increasing expression of eNOS independently
of sustained augmented gene transcription.
endothelium-dependent vasodilation; isolated rat lungs; endothelial nitric oxide synthase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PREMENOPAUSAL WOMEN
have a significantly lower risk of heart disease compared with
age-matched men and postmenopausal women (38). A portion
of the cardioprotective action of estrogen appears to be mediated by
actions on the vascular wall. This study and others have shown that
gender influences the incidence of cardiovascular disease and that this
gender effect appears to be mediated primarily by estrogen. A clinical
study (5) has shown that intracoronary infusions of
17-estradiol (E2
) increase endothelium-dependent relaxation to acetylcholine (ACh) in women compared with that in men.
In addition, Gilligan et al. (11) demonstrated that physiological doses of E2
administered in vivo enhanced
ACh vasorelaxation of large conductance and coronary resistance
arteries. Therefore, the beneficial effect of estrogen in the
vasculature appears to be due in part to upregulation of endothelial
nitric oxide (NO) production. This is supported by a study
(37) demonstrating that E2
increases the
activity of endothelial NO synthase (eNOS) in uterine artery, heart,
and skeletal muscle. Similarly, cultured human vascular endothelial
cells incubated for 24 h with E2
had increased
expression of eNOS that was dose dependent (16). In addition, E2
treatment increased eNOS mRNA levels in
skeletal muscle (37), suggesting that increases in NOS
activity are caused by the induction of gene transcription. Together,
these studies provide strong evidence that E2
enhances
NO-mediated vasodilation in systemic vascular beds.
Although many studies have explored the mechanisms of estrogen
modulation of vascular tone in the systemic circulation, estrogen regulation of vasomotor responses in the pulmonary circulation is less
clear. Early studies by Hultgren et al. (17) and Ergueta et al. (9) demonstrated that the incidence of chronic
mountain sickness and high-altitude pulmonary edema were significantly less in women compared with men. In addition, female rats exposed to
chronic hypoxia exhibited less pulmonary arterial hypertension (24) and attenuated right ventricular hypertrophy
(22) compared with age-matched males. Wetzel and Sylvester
(39) demonstrated that pulmonary vasomotor responses to
hypoxia were attenuated in isolated lungs from adult female sheep
compared with lungs from males. However, another study
(10) suggested that E2 augments vasoconstriction in pulmonary arteries, so the effect of
E2
on pulmonary vascular resistance is unclear.
Similar to the systemic circulation, one mechanism for estrogen
regulation of pulmonary vasodilation may be augmented by NO production.
For example, fetal pulmonary endothelial cells respond to estrogen by
increasing eNOS mRNA levels (20) and eNOS activity (33). Therefore, E2 may protect the
pulmonary vasculature during stresses such as hypoxia or lung disease
by increasing the synthesis and release of the potent vasodilator NO.
However, the magnitude and mechanisms of in vivo estrogen regulation of
eNOS in the pulmonary vasculature are not known. In addition, little is
known about in vivo E2
regulation of NO synthesis and
release in other vascular beds when E2
is maintained at
physiological rather than pharmacological levels. Therefore, these
experiments examined eNOS expression and activity in the systemic and
pulmonary vasculatures after E2
replacement therapy in
ovariectomized (OVX) female rats. We hypothesized that physiological
levels of E2
would upregulate eNOS expression, leading
to enhanced synthesis and release of NO in both the systemic and
pulmonary vasculatures.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All protocols and animal handling were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine.
Animal Preparation
Female Sprague-Dawley rats (250-300 g; Harlan Industries) were anesthetized with halothane, and the ovaries were removed bilaterally. After a 4-wk recovery to deplete endogenous E2Isolated Aortic Ring Tissue Bath Preparation
After 7 days of treatment, E2Isolated Aortic Ring Tissue Bath Protocol
Contractility studies determine if E2 treatment in
vivo enhances endothelium-dependent relaxation.
To determine whether chronic in vivo E2
treatment
enhances endothelium-dependent relaxation in the systemic circulation, we examined the vasodilatory responses to ACh in contracted aortic rings. Tissues were contracted with PE (10
6 mol/l), and
cumulative concentration-response curves to ACh (10
10 to
10
4.5 mol/l) were generated. Tissues were rinsed and
incubated with N
-nitro-L-arginine
(L-NNA; 100 µmol/l) for 30 min before relaxation was
again measured to evaluate ACh-stimulated NOS activity in vessels from
vehicle- and E2
-treated rats.
Isolated Perfused Lung Preparation
The isolated lung preparation was used to assess segmental vascular resistances in rat lungs as previously described by Eichinger and Walker (7). Briefly, lungs were isolated from OVX rats treated with E2Isolated Perfused Lung Protocol
Total, arterial, and venous vasodilatory responses to the
endothelium-dependent vasodilator ionomycin.
To determine if 7 days of in vivo treatment with E2
increased eNOS activity in pulmonary arteries, we examined total,
arterial, and venous vasodilatory responses to eNOS stimulation by
ionomycin (calcium ionophore) in lungs from E2
- and
vehicle-treated rats. Lungs were isolated and equilibrated as described
in Isolated Perfused Lung Preparation. After 30 min
of equilibration, lungs were constricted with the thromboxane mimetic
U-46619 to elicit a stable pressor response of ~10 Torr above
baseline pressure. Lungs that required a dose of U-46619 >8 µg to
elicit a 10 Torr increase in tone were eliminated. Once
vasoconstriction was stable, NO-dependent vasodilation was measured
with ionomycin (1 µmol/l). Pulmonary capillary pressure (Pcap) was
estimated with the double-occlusion technique (7, 13, 36).
Total and segmental pulmonary vascular resistances were calculated
during basal, constricted, and dilated conditions with the equations
RT = (Pa
Pv)/Q, Ra = (Pa
Pcap)/Q, and
Rv = (Pcap
Pv)/Q, where RT is total
resistance, Ra is arterial resistance, Rv is venous resistance, and Q
is flow. Vasodilatory responses were calculated as percent reversal of
U-46619-induced vasoconstriction for total, arterial, and venous segments.
Western Blot Analysis
eNOS expression in aortas and lung homogenates from
E2- and vehicle-treated rats.
eNOS protein content was evaluated with standard immunoblotting
methods. The left lobes of the lungs and the thoracic aortas from
E2
- and vehicle-treated groups were rapidly isolated,
rinsed briefly in ice-cold homogenizing buffer, and frozen in liquid nitrogen. Frozen tissue was coarsely ground in liquid nitrogen with a
precooled mortar and pestle and then further homogenized in a glass
dounce homogenizer in ice-cold Tris · HCl buffer containing EDTA (0.3 mg/ml), leupeptin (5 µl/ml), pepstatin A (0.7 µg/ml), aprotinin (2 µg/ml), and phenylmethylsulfonyl fluoride (20 µg/ml). Homogenates were spun at 800 g at 4°C for 1.5 min to
remove cellular debris, the supernatant was drawn off, and an aliquot
was analyzed for protein concentration with the Bradford method
(Bio-Rad protein assay). Samples were dissolved in 5× sample buffer (1 µg/µl), boiled for 5 min, and separated in 15% polyacrylamide
gels. In addition to E2
and vehicle samples (10 µg/lane, aorta; 30 µg/lane, lung), each gel contained molecular
weight markers and an eNOS standard (Transduction Laboratories).
Separated proteins were transferred to polyvinylidene difluoride
membranes, blocked overnight, and probed with a monoclonal antibody
specific for eNOS (1:1,000; Transduction Laboratories). Enhanced
chemiluminescence development in conjunction with a horseradish
peroxidase-labeled secondary antibody (1:5,000) was used to visualize
eNOS protein. The relative quantity of protein was determined with
SigmaGel software (SPSS). The molecular weight markers and an eNOS
protein standard were used to verify antibody detection of proteins of
the expected size and to normalize values between gels. In a separate
experiment, 5, 10, and 15 µg/lane of protein were analyzed to ensure linearity.
Immunohistochemistry
Immunostaining for eNOS in aortas from E2- and
vehicle-treated rats.
To further establish if E2
increases eNOS, we assessed
eNOS immunostaining in aortas and pulmonary arteries. Cleaned aortic segments were placed in specimen molds that contained embedding medium
(Tissue-Tek O.C.T. compound) and frozen in isobutane cooled with liquid nitrogen. Transverse sections of the vessels were cut (10 µm) and thaw-mounted on glass slides (Superfrost Plus). Sections were
treated with 0.33% hydrogen peroxide to inhibit endogenous
peroxidases, blocked with buffer containing normal horse serum (4%),
and incubated with a mouse monoclonal antibody against eNOS (1:2,500;
Transduction Laboratories) for 24 h at 4°C. A rat-absorbed,
biotinylated horse anti-mouse IgG secondary antibody (1:400; Vector
Laboratories) was used in combination with the peroxidase substrate
3,3'-diaminobenzidine tetrahydrochloride dihydrate (0.07%) in hydrogen
peroxide (0.002%). Nonspecific binding was evaluated by substituting
total mouse IgG for the primary antibody. Densitometric analysis was
performed with a Bioquant image analysis system with green wavelength
illumination (×40 objective). Densitometry units were expressed as
gray levels (0 = 100% light transmittance and 225 = 0%
light transmittance). A densitized image was generated after
subtracting out background, and densitometry values were calculated
from the average density of all regions that stained above threshold.
Immunohistochemical staining for eNOS in lungs from
E2- and vehicle-treated rats.
Lungs were prepared for immunohistochemical analysis as described
previously (26). After isolation, lungs were perfused with
PSS containing 4% bovine serum albumin and papaverine
(10
4 mmol/l) followed by perfusion with phosphate buffer
containing paraformaldehyde (5%), glutaraldehyde (0.1%), and
papaverine (10
4 mmol/l). During perfusion with PSS and
fixatives, Pv was maintained at 12 Torr to elicit maximal recruitment
(8). Lungs were inflated, fixed, and embedded in optimum
cutting temperature compound. Transverse sections were cut (10 µm)
and thaw-mounted onto glass slides (Superfrost Plus). Lung sections
were immunostained following the same protocol described for aortic
tissues (see Immunostaining for eNOS in aortas from
E2
- and vehicle-treated rats). Serial sections were
stained for elastin and counterstained with Van Gieson solution
(Accustain Elastic Stain; Sigma) to identify arteries and veins.
Arteries were identified by the presence of an internal elastic lamina and ranged in size from 200 to 300 µm. Densitometry was performed as
described in Immunostaining for eNOS in aortas from
E2
- and vehicle-treated rats.
Ribonuclease Protection Assay
Construction of probe templates for ribonuclease protection assay
analysis.
Probe templates for ribonuclease protection assays (RPAs) were
constructed for rat eNOS and malate dehydrogenase (MDH). Rat cDNA was
synthesized from rat lung with reverse transcription. Segments of the
cDNA obtained were amplified for eNOS and MDH with PCR, and primer
sequences were made with archived rat and mouse DNA sequences
(GenBank). Probe templates were amplified with Pyrococcus
furiosus DNA polymerase (Stratagene), and the primer sequences are
listed in Table 1. The eNOS PCR product was inserted into the SrfI site of the pPCR-Script
Amp SK(+) cloning vector (GenBank accession no. U46017) with the
PCR-Script Amp cloning kit (Stratagene). Plasmid DNA was prepared from
transformants with the QIAprep Spin miniprep kit (QIAGEN) and subjected
to restriction analysis to determine the orientation of inserts
relative to the vectors T7 and T3 RNA polymerase promoters. eNOS and
MDH PCR templates were prepared with reverse PCR primers with a T7 RNA
polymerase promoter sequence, 5'-TAATACGACTCACTATAGGGAGGA-3', appended
to the 5'-end of the original reverse primer. The new primer was used
to reamplify the original PCR products, resulting in the T7 promoter
being expressed upstream of the antisense RNA strand. Probe templates
were purified with the QIAquick PCR purification kit (QIAGEN). A
MAXIscript in vitro transcription kit (Ambion) was used according to
the manufacturer's recommendations to prepare the radiolabeled
antisense cRNA. Full-length RNA probes were purified by PAGE on 5%
Tris-borate-EDTA (TBE)-urea gels, and the amount of radioactive label
was determined by scintillation counting. The eluted probes were stored
in elution buffer at 20°C until needed.
|
eNOS mRNA expression in thoracic aortas and lungs from
E2- and vehicle-treated rats.
Total RNA was isolated from lungs and aortas by homogenization in
TRIzol reagent (GIBCO BRL). A RPA Kit II (Ambion) was used to
quantitate mRNA in homogenates from treated and control groups according to the manufacturer's recommendations. In brief, aliquots of
the labeled probe containing 2-8 × 104
counts/min (cpm) were added to 5 µg of total RNA. Samples were precipitated in ethanol, resuspended in hybridization buffer, and
heated to 95°C for 4 min. The hybridization reactions were incubated
overnight at 42°C, then digested with RNase A (2.5 U/ml), and cloned
with RNase T1 (100 U/ml). Digested products were precipitated, resuspended in 4-6 µl of formamide loading buffer, loaded onto 5% TBE-urea polyacrylamide minigels (Bio-Rad), and electrophoresed. Dried gels were exposed to a phosphor storage screen (Molecular Dynamics) and scanned with a STORM 860 phosphorimager (Molecular Dynamics). Bands were quantitated with ImageQuant software, and products are expressed as a percent of the internal control gene MDH.
Additional hybridizations containing 5, 10, and 15 µg of total RNA
from lung tissue were performed to ensure linearity.
Drugs and Chemical Solutions
Indomethacin (Sigma) was dissolved in ethanol. PE, ACh, and L-NNA (all from Sigma) were dissolved in double-distilled water (ddH2O). Ionomycin (Calbiochem) was diluted in dimethyl sulfoxide (Sigma) and stored at 4°C. Meclofenamate (Sigma) was prepared in normal saline. U-46619 was diluted in 95% ethanol and stored atStatistics
Data are reported as means ± SE, with P ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasma E2 Concentrations in OVX Female Rats
Effects of E2 In Vivo on Endothelium-Dependent
Relaxation Induced by ACh in Aortic Rings
|
Because E2 administration enhanced endothelium-dependent
vasodilation in aortic rings, vasodilatory responses to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were
assessed to determine if ACh-enhanced relaxation in tissues from
E2
-treated animals was due to increased vascular smooth
muscle (VSM) reactivity to NO. Relaxation to SNAP was not
different between groups, suggesting that enhanced relaxation to ACh in
tissues from the E2
-treated animals was not due to
changes in VSM sensitivity to NO (Fig. 1B). Therefore, these
data suggest that ACh-induced relaxation is NO dependent and
selectively augmented in aorta by E2
treatment of OVX rats.
Effects of E2 Administration In Vivo on
Endothelium-Dependent Dilation in Perfused Isolated Lungs
|
|
Effects of eNOS Inhibition by L-NNA on Augmented Endothelium-Dependent Dilation
To determine whether the augmented pulmonary vasodilatory response to ionomycin in lungs from E2
|
Western Blot Analysis of Endothelial Expression of eNOS in Aortas
and Lungs From E2- and Vehicle-Treated OVX Rats
|
Because lungs from E2-treated animals exhibited enhanced
vasodilation to ionomycin and this response was abolished by
L-NNA, eNOS protein levels were also evaluated in lung
tissue homogenates (Fig. 3B). However, there was no
difference in eNOS protein expression between groups as evaluated by
Western analysis.
Immunohistochemistry for eNOS in Aortas and Lungs From
E2- and Vehicle-Treated OVX Rats
|
Analysis of eNOS mRNA Levels in Aortas and Lungs From
E2- and Vehicle-Treated OVX Rats
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been suggested that estrogen directly modulates vascular
tone by enhancing endothelium-dependent vasodilation. We observed that
physiological levels of E2 replacement in OVX
rats augmented endothelium-dependent vasodilation and enhanced
eNOS activity in aortas and pulmonary blood vessels. The
observation that E2
treatment in vivo increased
endothelium-dependent relaxation in the aorta is consistent with
results from previous studies with isolated uterine arteries from
guinea pigs (4) and femoral arteries from rabbits
(12). Our results in the pulmonary circulation demonstrate
for the first time that endothelium-dependent vasodilation in the lung
is enhanced by chronic E2
treatment. These results expand the role for E2
in protecting cardiovascular function.
The mechanisms by which E2 increases
endothelium-dependent relaxation have not been clearly elucidated.
Some studies suggest that E2
in vivo selectively
enhances NO-dependent relaxation. For example, Cheng et al.
(3) observed that aortic rings from rats chronically
treated with E2
demonstrated enhanced relaxation to
ACh but not to histamine, an endothelium-dependent vasodilator that
activates phospholipase A2 production of prostacyclin. This indirectly suggested that long-term administration of E2
enhances endothelium-dependent relaxation of blood vessels by
upregulating eNOS. Our direct measures of eNOS protein provide
additional evidence for eNOS upregulation by E2
.
Reports of estrogen actions in other vascular beds have demonstrated
that E2 relaxes uterine, skin, and coronary vessels (28, 31, 40). During pregnancy, when circulating levels of
estrogen are high, pulmonary vasoconstriction to hypoxia and angiotensin II is decreased (23, 34). Moreover, hypoxic
pulmonary vasoconstriction in lungs isolated from female sheep is less
compared with constriction in lungs from males (39).
Together, these studies provide indirect evidence that estrogen
enhances vasodilation in multiple vascular beds including the pulmonary
circulation. Thus we evaluated chronic E2
treatment on
vasoreactivity and eNOS expression in both the aorta and the pulmonary
vasculature. We hypothesized that if E2
upregulates eNOS
expression, then endothelium-dependent vasodilators would elicit
greater NO-dependent relaxation in arteries from
E2
-treated animals.
In support of this hypothesis, we found that lungs isolated from
E2-treated rats exhibited augmented vasodilatory
responses to ionomycin and that aortas from E2
-treated
rats had augmented endothelium-dependent relaxation. Because
E2
elicits an endothelium-dependent cardioprotective
effect in the systemic and coronary vasculatures, these results suggest
it does the same in the pulmonary vasculature. In this way,
E2
may maintain low pulmonary vascular resistance in
times of stress such as lung disease and hypoxia.
Previous studies have demonstrated gender differences in hypoxic
pulmonary hypertension (24) and in hypoxia-induced
pulmonary vascular remodeling (39). Although our study did
not investigate the role of E2 during hypoxia, it did
study the mechanisms associated with E2
regulation of
pulmonary endothelial function, which is modified by hypoxia. We found
that NOS inhibition with L-NNA attenuated ionomycin-induced
vasodilation in lungs from both vehicle- and E2
-treated
animals and that this treatment abolished the enhanced vasodilation in
lungs from E2
-treated rats. Although the ionomycin vasodilatory response was not completely abolished by NOS inhibition, there was no difference in vasodilation between groups when NOS activity was inhibited, suggesting that E2
enhances
pulmonary endothelial cell NOS activity.
In aortic studies, the augmented component of the ACh-induced aortic
relaxation by E2 was also completely abolished by NOS inhibition. This is in agreement with the observation (19)
that E2
enhances flow-induced dilation in the brachial
arteries of postmenopausal women only in the absence of a NOS
antagonist. Together, these observations show that the enhanced
endothelium-dependent relaxation that follows E2
treatment requires NOS.
Interestingly, Chang et al. (2) demonstrated that
E2 may stimulate prostacyclin synthesis in VSM. Like NO,
prostacyclin is a potent pulmonary vasodilator capable of contributing
to the enhanced vasodilatory effects that follow E2
treatment. However, in our preparation, prostacyclin is unlikely to
have contributed to either the pulmonary dilation or the augmented
aortic response because all preparations were treated with a
cyclooxygenase inhibitor. In the isolated lungs, the dilation
remaining after meclofenamate and L-NNA treatment
thus appeared to be dependent on other endothelium-derived factors such
as endothelium-dependent hyperpolarizing factor. Therefore, because the
difference in the vasodilatory response to ionomycin between lungs from
E2
- and vehicle-treated rats was abolished by
L-NNA treatment but was present during cyclooxygenase inhibition and all aortic relaxation was abolished by
L-NNA, we concluded that the enhanced vasodilation induced
by estrogen replacement was due to enhanced NO synthesis.
In addition to affecting endothelial function, E2 may
further enhance vasodilation by altering VSM sensitivity to NO. In postmenopausal women, short-term administration of E2
augments vasodilatory responses to ACh in the coronary (11,
25) and systemic (35) circulations. These studies
showed that E2
improved both endothelium-dependent (ACh)
and -independent (sodium nitroprusside) vasodilatory responses in
coronary and forearm blood vessels, suggesting that E2
can influence both endothelial and VSM function. In contrast to what
was shown in postmenopausal women but in agreement with animal studies
(3), we observed no differences in sensitivity to the NO
donor SNAP in denuded aortic rings from E2
- and
vehicle-treated rats. In addition, pulmonary vasodilation to NO donors
was not augmented by E2
treatment (Resta TC and Walker
BR, unpublished data). Therefore, our data cannot be explained
by enhanced VSM sensitivity to NO.
Many substances, including E2, can regulate eNOS
activity acutely by increasing endothelial cell intracellular calcium
concentration ([Ca2+]i) to activate eNOS and
by stimulating NO release. However, the mechanism of the chronic effect
of E2
upregulation of NOS activity is less clear.
Classically, the regulation of transcription by E2
is
mediated by receptor binding and transport to the nucleus. The
subsequent regulation of gene transcription and protein expression is
dependent on the presence of an estrogen response element, which is the
binding site for the estrogen receptor complex. It has been suggested
that the eNOS promoter contains an estrogen response element as well as
an activator protein-1 binding site, both of which are thought to
regulate enzyme expression and NO release (21). Although a
functional study (16) showed E2
upregulation of eNOS (16), an electromobility shift study
(21) found that the estrogen receptor does not bind to the
NOS promoter. Therefore, E2
regulation of eNOS
expression remains controversial. With Western analysis, we found that
E2
treatment in vivo increased eNOS protein levels in
rat aorta compared with vehicle treatment. We further demonstrated that
eNOS-immunoreactive staining was more intense in aortas from
E2
-treated rats. These findings are in agreement with
our observation that ACh relaxation was augmented in the aorta and with
previous reports (16, 29, 37) that demonstrated increased
eNOS protein expression after chronic administration of higher doses of
E2
.
To date, no studies have investigated the effect of E2
treatment in vivo on NOS expression in the pulmonary vasculature. However, an in vitro study (20) has demonstrated that
chronic E2
administration upregulates eNOS gene
expression in fetal pulmonary arterial endothelial cells. Thus this
study suggests that E2
may augment pulmonary
vasodilation by upregulating the NO-generating enzyme eNOS.
We found that E2 treatment increased eNOS immunostaining
in pulmonary arteries. Arterial and venous morphology, determined in
serial sections, made it apparent that eNOS staining was enhanced only
in arteries. This was in agreement with our functional studies that
showed that only pulmonary arterial vasodilation was enhanced in
E2
-treated rats. In addition to immunohistochemical
analysis, Western blots were used to quantitate eNOS protein
expression. In contrast to immunohistochemical analysis, we found no
detectable differences in eNOS expression in lung homogenates from the
E2
- and vehicle-treated groups with this method. One
possibility for the disparate findings with these two techniques is
that the level of E2
induction within the lung is
localized within the arterial vascular endothelium and is too small to
be detected by Western analysis of whole lung homogenates.
Because eNOS protein levels in E2-treated rats appeared
to be enhanced in both vascular beds, we also examined eNOS mRNA levels
with a RPA. In contrast to eNOS immunostaining, we found no increases
in mRNA in either aorta or lung from rats treated with
E2
. It is interesting that other studies (14,
32) reporting increased eNOS protein levels after
E2
administration have not detected increased message
either. The increase in eNOS expression apparent in immunostained
sections suggests that the augmented endothelium-dependent responses
associated with E2
treatment are due to elevated NOS
expression but not to persistent increases in eNOS message.
In conclusion, replacing E2 in OVX rats to produce
physiological plasma levels enhances endothelium-dependent relaxation and increases eNOS expression in both thoracic aortas and pulmonary arteries. Thus it is likely that the observed augmentation of the
endothelium-dependent vasodilation in the aortas and lungs from
E2
-treated female rats is due to increased levels of the NO-generating enzyme eNOS. Enhanced eNOS levels may thus contribute to
E2
cardioprotective effects in both systemic and
pulmonary arteries.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Pam Allgood and Scott Early for technical assistance in this study.
![]() |
FOOTNOTES |
---|
This work was supported by an American Physiological Society Predoctoral Porter Fellowship (to R. Gonzales), National Heart, Lung, and Blood Institute Grant HL-03852 (to N. Kanagy), and an American Heart Association Scientist Development Grant (to N. Kanagy).
Address for reprint requests and other correspondence: R. Gonzales, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: rgonzales{at}salud.unm.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 18 July 2000; accepted in final form 19 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Butcher, RL,
Collins WE,
and
Fugo NW.
Plasma concentrations of LH, FSH, prolactin, progesterone, and estradiol-17 throughout the 4-day estrous cycle of the rat.
Endocrinology
94:
1704-1708,
1978[ISI][Medline].
2.
Chang, W,
Nakao J,
Orimo H,
and
Murota S.
Stimulation of prostaglandin cyclooxygenase and prostacyclin synthetase activities by estradiol in rat aortic smooth muscle cells.
Biochim Biophys Acta
620:
472-482,
1980[ISI][Medline].
3.
Cheng, DY,
Feng CJ,
Kadowitz PJ,
and
Gruetter CA.
Effects of 17-estradiol on endothelium-dependent relaxation induced by acetylcholine in female rat aorta.
Life Sci
55:
187-191,
1994.
4.
Cheng, DY,
and
Gruetter CA.
Chronic estrogen alters contractile responsiveness to angiotensin II or norepinephrine in female rat aorta.
Eur J Pharmacol
5:
97-102,
1992.
5.
Collins, P,
Rosano GM,
Sarrel PM,
Ulrich L,
Adamopoulos S,
Beale CM,
McNeil JG,
and
Poole-Wilson A.
17-Estradiol attenuates acetylcholine-induced constriction in women but not men with coronary heart disease.
Circulation
92:
24-30,
1993
6.
Dawson, CA,
Linehan JH,
and
Rickaby DA.
Pulmonary microcirculatory hemodynamics.
Ann NY Acad Sci
384:
90-106,
1982[ISI][Medline].
7.
Eichinger, MR,
and
Walker BR.
Enhanced pulmonary arterial dilation to arginine vasopressin in chronically hypoxic rats.
Am J Physiol Heart Circ Physiol
267:
H2413-H2419,
1994
8.
Eichinger, MR,
and
Walker BR.
Nitric oxide and cGMP do not affect fluid flux in isolated rat lungs.
J Appl Physiol
80:
69-76,
1997
9.
Ergueta, J,
Spielvogel H,
and
Cudkowicz L.
Cardio-respiratory studies in chronic mountain sickness (Monge's syndrome).
Respiration
28:
485-517,
1971[ISI][Medline].
10.
Farhat, MY,
and
Ramwell PW.
Estradiol potentiates the vasopressor response of the isolated perfused rat lung to the thromboxane mimic U-46619.
J Pharmacol Exp Ther
261:
686-691,
1992[Abstract].
11.
Gilligan, DM,
Quyyumi AA,
and
Cannon RO.
Effects of physiological levels of estrogen on coronary vasomotor function in post-menopausal women.
Circulation
89:
2545-2551,
1994[Abstract].
12.
Gisclard, V,
Miller VM,
and
Vanhoutte PM.
Effect of 17-estradiol on endothelium-dependent responses in the rabbit.
J Pharmacol Exp Ther
244:
19-23,
1988[Abstract].
13.
Hakim, TS.
Flow-induced release of EDRF in the pulmonary vasculature: site of release and action.
Am J Physiol Heart Circ Physiol
267:
H363-H369,
1994
14.
Hassen, SS,
Ohara Y,
Navas JP,
Peterson TE,
Dockery S,
and
Harrison DG.
Endothelial nitric oxide synthase regulation by estrogens (Abstract).
Circulation
88:
I80,
1993.
15.
Hayashi, T,
Fukuto JM,
Ignarro LJ,
and
Chaudhuri G.
Basal release of nitric oxide from aortic rings is greater in female rabbits that in male rabbits.
Proc Natl Acad Sci USA
89:
11259-11263,
1992[Abstract].
16.
Hishikawa, K,
Nakaki T,
Marumo T,
Suzuki H,
Kato R,
and
Saruta T.
Up-regulation of nitric oxide synthases by estradiol in human aortic endothelial cells.
FEBS Lett
360:
291-293,
1995[ISI][Medline].
17.
Hultgren, HN,
Lopez C,
Lundberge E,
and
Miller H.
Physiologic studies of pulmonary edema at high altitude.
Circulation
29:
393-408,
1964[ISI].
18.
Legan, SJ,
Coon GA,
and
Karsch FJ.
Role of estrogen as initiator of daily LH surges I ovariectomized rats.
Endocrinology
96:
50-56,
1975[ISI][Medline].
19.
Lieberman, EH,
Gerhard MD,
Uheta A,
Selwyn AP,
Yeung AC,
Ganz P,
Anderson TJ,
and
Creager MA.
Flow induced vasodilation of the human brachial artery is impaired in patients <40 years of age with coronary artery disease.
Am J Cardiol
78:
1210-1214,
1996[ISI][Medline].
20.
MacRitchie, AN,
Jun SS,
Chen Z,
German Z,
Yuhanna IS,
Sherman TS,
and
Shaul PW.
Estrogen upregulation of endothelial nitric oxide gene expression in fetal pulmonary artery endothelial cells.
Circ Res
81:
355-362,
1997
21.
Marsden, PA,
Heng HH,
Scherer SW,
Stewart RJ,
Hal AV,
Shi XM,
Tsui L,
and
Schappert KT.
Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase.
J Biol Chem
268:
17478-17488,
1993
22.
McMurtry, IF,
Frith CH,
and
Will DH.
Cardiopulmonary responses of male and female swine to simulated high altitude.
J Appl Physiol
35:
459-462,
1973
23.
Moore, LG,
and
Reeves JT.
Pregnancy blunts pulmonary vascular reactivity in dogs.
Am J Physiol Heart Circ Physiol
239:
H297-H301,
1980[ISI][Medline].
24.
Rabinovitch, M,
Gamble WJ,
Miettinen O,
and
Reid L.
Age and sex influence on pulmonary hypertension of chronic hypoxia and recovery.
Am J Physiol Heart Circ Physiol
240:
H62-H72,
1981
25.
Reis, SE,
Gloth ST,
and
Blumenthal RS.
Ethinyl estradiol acutely potentiates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women.
Circulation
89:
52-60,
1994[Abstract].
26.
Resta, TC,
Gonzales RG,
Dail WG,
Sanders TC,
and
Walker BR.
Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension.
Am J Physiol Heart Circ Physiol
272:
H806-H813,
1997
27.
Resta, TC,
and
Walker BR.
Chronic hypoxic selectivity augments endothelium-dependent pulmonary arterial dilation.
Am J Physiol Heart Circ Physiol
270:
H888-H896,
1996
28.
Rosenfeld, CR,
and
Rivera R.
Circulatory response to systemic infusions of estrone and estradiol 17 alpha in non-pregnant oophorectomized ewes.
Am J Obstet Gynecol
132:
442-448,
1978[ISI][Medline].
29.
Rosseli, M,
Imthum B,
Keller PJ,
Jackson EK,
and
Dubey RK.
Circulating nitric oxide (nitrate/nitrite) levels in postmenopausal women substituted with 17-estradiol and norethisterone acetate: a two year follow-up study.
Hypertension
25:
848-853,
1995
30.
Rubin, BS,
Mitchell S,
Lee EC,
and
King JC.
Reconstruction of populations of luteinizing hormone releasing hormone neurons in young and middle-aged rats reveal progressive increases in subgroups expressing Fos protein on proestrus and age-related deficits.
Endocrinology
136:
3823-3830,
1995[Abstract].
31.
Salas, E,
Lopez Villarroya M,
Sanchez-Garcia P,
DePascual R,
Dixon WR,
and
Garcia AG.
Endothelium-independent relaxation by 17-alpha-estradiol of pig coronary arteries.
Eur J Pharmacol
258:
47-55,
1994[ISI][Medline].
32.
Schray-Utz, B,
Zeiher AM,
and
Busse R.
Expression of constitutive nitric oxide synthase in cultured endothelial cells is enhanced by 17-estradiol (Abstract).
Circulation
88:
I80,
1993.
33.
Shaul, PW.
Rapid activation of endothelial nitric oxide synthase by estrogen.
Steroids
64:
28-34,
1999[ISI][Medline].
34.
Sylvester, JT,
Gordon JB,
Malamet RL,
and
Wetzel RC.
Prostaglandins and estrogen-induced attenuation of hypoxic pulmonary vasoconstriction.
Chest
88:
252S-254S,
1985[Abstract].
35.
Tagawa, H,
Shimokawa H,
Tagawa T,
Matsumoto M,
Hirooka Y,
and
Takeshita A.
Short-term estrogen augments both nitric oxide-mediated and non-nitric oxide-mediated endothelium-dependent forearm vasodilation in postmenopausal women.
J Cardiovasc Pharmacol
30:
481-487,
1997[ISI][Medline].
36.
Townsley, MI,
Korthuis RJ,
Rippe B,
Parker JC,
and
Taylor AE.
Validation of double vascular double occlusion method for Pc,i in lung and skeletal muscle.
J Appl Physiol
61:
127-32,
1986
37.
Weiner, CP,
Lizasoain I,
Baylis SA,
Knowles RG,
Charles IG,
and
Monocada S.
Induction of calcium-dependent nitric oxide synthases by sex hormones.
Proc Natl Acad Sci USA
91:
5212-5216,
1994[Abstract].
38.
Weiss, NS.
Relationship of menopause to serum cholesterol and arterial blood pressure: the United States health examination survey of adults.
Am J Epidemiol
96:
237-241,
1972[ISI][Medline].
39.
Wetzel, RC,
and
Sylvester JT.
Gender differences in hypoxic vascular response of isolated sheep lungs.
J Appl Physiol
55:
100-104,
1983
40.
Williams, JK,
Adams MR,
and
Klopfenstein HS.
Estrogen modulates responses of athrosclerotic coronary arteries.
Circulation
81:
1680-1687,
1990[Abstract].