Vascular Physiology Group, Department of Cell Biology and Physiology, Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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Female rats develop
less severe pulmonary hypertension (PH) in response to chronic hypoxia
compared with males, thus implicating a potential role for ovarian
hormones in mediating this gender difference. Considering that estrogen
upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic
vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by
increasing eNOS expression and activity. To test this hypothesis, we
examined responses to the endothelium-derived NO-dependent dilator
ionomycin and the NO donors
S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from
the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries,
3) CH ovariectomized rats given 17-estradiol
(E2
), and 4) CH ovariectomized rats given
vehicle. Additional experiments assessed pulmonary eNOS levels in each
group by Western blotting. Our findings indicate that E2
attenuated chronic hypoxia-induced right ventricular hypertrophy,
pulmonary arterial remodeling, and polycythemia. Furthermore, although
CH augmented vasodilatory responsiveness to ionomycin and increased
pulmonary eNOS expression, these responses were not potentiated by
E2
. Finally, responses to
S-nitroso-N-acetylpenicillamine and spermine
NONOate were similarly attenuated in all CH groups compared with
normoxic control groups. We conclude that the inhibitory influence of
E2
on chronic hypoxia-induced PH is not associated with
increased eNOS expression or activity.
chronic hypoxia; right ventricular hypertrophy; vascular remodeling; nitric oxide-dependent vasodilation; endothelial nitric oxide synthase
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INTRODUCTION |
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PULMONARY
HYPERTENSION associated with chronic hypoxia is characteristic of
chronic obstructive pulmonary diseases as well as of residence at high
altitude. It has been noted that women with chronic obstructive
pulmonary diseases exhibit a decreased risk of mortality compared with
men (36), although insufficient comparisons have been made
to determine the etiology of this sexual dimorphism. Consistent with
this observation are studies (22, 28) indicating that
female rats and swine develop less severe pulmonary hypertension, right
ventricular hypertrophy, arterial remodeling, and polycythemia in
response to chronic hypoxia compared with males. Isolated ovine lung
studies (15, 38) have further demonstrated inhibitory
effects of 17-estradiol (E2
) on hypoxic pulmonary
vasoconstriction (HPV). These findings are consistent with a role for
estrogen in attenuating both fixed (remodeling, polycythemia) and
active (HPV) components of chronic hypoxia-induced pulmonary
hypertension, although the mechanisms by which estrogen may exert such
protective influences have not been examined.
Studies in other vascular beds suggest estrogen exhibits
antiatherogenic properties that are mediated in part by alterations in
endothelial function (1; reviewed in Ref. 17), including enhanced synthesis of the vasodilator and antimitogenic factor nitric
oxide (NO) (4, 17, 18, 21). Sustained increases in NO
synthesis in response to estrogen have been attributed to upregulation
of endothelial NO synthase (eNOS) in both systemic and pulmonary
vascular tissue (17, 18, 21). Consistent with these
findings are preliminary reports from our laboratory suggesting that 1 wk of estrogen administration to normoxic ovariectomized (OVX) rats
augments endothelium-derived NO (EDNO)-dependent pulmonary vasodilation
(14) and elevates pulmonary vascular eNOS levels (13). We therefore hypothesized that the inhibitory
influence of estrogen on the development of chronic hypoxia-induced
pulmonary hypertension is a function of increased eNOS expression and
activity. eNOS activity was assessed by examining responses to the
EDNO-dependent vasodilator ionomycin in lungs isolated from chronically
hypoxic (CH) rats with intact ovaries as well as from CH OVX rats
receiving E2 or vehicle for the duration of hypoxic
exposure. Pulmonary eNOS expression was determined for each group of
rats by Western blotting. Our findings indicate that although
E2
attenuates chronic hypoxia-induced right ventricular
hypertrophy and pulmonary arterial remodeling, this protective
influence of E2
is not associated with increased
pulmonary eNOS expression or enhanced EDNO-dependent responsiveness.
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METHODS |
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All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM).
Experimental Groups
Four groups of female Sprague-Dawley rats (200-350 g; Harlan Industries) were used for all experiments: 1) normoxic rats with intact ovaries, 2) CH rats with intact ovaries, 3) CH OVX rats that received E2Surgical Procedures for Ovariectomy and Osmotic Pump Implantation
Rats designated for ovariectomy were anesthetized with a mixture of ketamine (90 mg/kg im) and acepromazine (0.9 mg/kg im). With sterile technique, ovaries were resected through bilateral flank incisions. Rats were allowed 2 wk to recover before implantation of the osmotic pumps (Alzet model 2ML4) for the 4-wk administration of E2Assessment of Plasma E2, Uterine Weight,
Polycythemia, and Right Ventricular Hypertrophy
Right ventricular hypertrophy was assessed as an index of chronic hypoxia-induced pulmonary hypertension with previously described methods (29-31). Briefly, after isolation of the heart, the atria and major vessels were removed from the ventricles. The right ventricle (RV) was dissected from the left ventricle and septum (LV+S), and each was weighed. The degree of right ventricular hypertrophy is expressed as the ratio of RV to total ventricle weight (T).
Vascular Morphometry
To determine whether the inhibitory influence of estrogen on chronic hypoxia-induced pulmonary hypertension was associated with decreased arterial remodeling, quantitative morphometric analyses of arterial cross sections were performed on lungs from each group of rats as described previously by Resta and colleagues (29, 32). Rats from each group were anesthetized with intraperitoneal pentobarbital sodium (25 mg). After the trachea was cannulated with a 17-gauge needle stub, the lungs were ventilated with a Harvard positive-pressure rodent ventilator (model 683) at a frequency of 55 breaths/min and a tidal volume of 2.5 ml with a warmed and humidified gas mixture (6% CO2 in room air). Inspiratory pressure was set at 9 cmH2O, and positive end-expiratory pressure was set at 3 cmH2O. After a median sternotomy, heparin (100 U) was injected directly into the RV, and the pulmonary artery was cannulated with a 13-gauge needle stub. The preparation was immediately perfused at 0.8 ml/min with a Masterflex microprocessor pump drive (model 7524-10) with a physiological saline solution (PSS) containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose with 4% (wt/vol) albumin, all from Sigma. Papaverine (10Isolated Lung Preparation
Separate sets of animals from each group were anesthetized with intraperitoneal pentobarbital sodium (25 mg). Lungs were isolated and perfused as described in Vascular Morphometry with the exceptions that papaverine was not included in the PSS and meclofenamate (10 µg/ml) was added to minimize the potential complicating influences of prostaglandins on vascular reactivity. This dose of meclofenamate is approximately threefold higher than that previously shown to provide effective inhibition of prostaglandin synthesis in this preparation (11). The perfusion rate was gradually increased to 30 ml · minIsolated Lung Protocols
Segmental vasodilatory responses to ionomycin.
To examine whether estrogen increases pulmonary eNOS activity, we
assessed total, arterial, and venous vasodilatory responses to the
non-receptor-mediated, EDNO-dependent vasodilator ionomycin (calcium
ionophore; Calbiochem) (33) in lungs from each group of
rats. Lungs were isolated and allowed to equilibrate as described in
Isolated Lung Preparation. Baseline capillary
pressure (Pc) was assessed by a double-occlusion procedure
at the end of the 30-min equilibration period to allow calculation of
segmental resistances (see Calculations and Statistics) as
described previously (29-33). After assessment of
baseline Pc, the thromboxane analog 9,11-dideoxy-9,11
-epoxymethanoprostaglandin F2
(U-46619; Cayman Chemical) was added to the perfusate reservoir until a stable arterial pressor response of ~10 mmHg was achieved. U-46619 provides consistent and stable pressor responses in this preparation and, unlike hypoxia, constricts both arterial and venous segments of
the pulmonary vasculature (29-33). Pc was
assessed at the plateau of the pressor response by double occlusion.
The vasculature was then dilated with 350 nM ionomycin, and
Pc was again determined at the point of maximal
vasodilation. Ionomycin was chosen as a non-receptor-mediated,
EDNO-dependent vasodilator in this study because interpretation of
responses to receptor-mediated agonists would be complicated by
possible changes in the number or affinity of their respective
receptors in response to hypoxia, ovariectomy, or E2
replacement. Furthermore, our laboratory has shown that this
concentration of ionomycin elicits submaximal vasodilatory responses in
this preparation (33) that correlate in magnitude with
vascular eNOS protein levels (29).
Inhibition of responses to ionomycin by
N-nitro-L-arginine.
The following protocols were employed to document the contribution of
endogenous NO in mediating vasodilatory responses to ionomycin in lungs
from each group of rats. Lungs were isolated as described in
Isolated Lung Preparation and perfused with PSS containing
300 µM N
-nitro-L-arginine
(L-NNA; Sigma). Resta and Walker (33) have previously demonstrated that this dose of L-NNA is
effective in inhibiting EDNO-dependent pulmonary vasodilation in the
isolated perfused rat lung. Segmental vasodilatory responses to
ionomycin (350 nM) were assessed after preconstriction with U-46619 as
described in Segmental vasodilatory responses to ionomycin.
Segmental vasodilatory responses to exogenous NO. Because any differences in reactivity to ionomycin between groups could potentially result from altered vascular smooth muscle reactivity to NO, additional experiments examined segmental responses to the NO donors S-nitroso-N-acetylpenicillamine (SNAP; 1 µM; Sigma) and spermine NONOate (2 µM; Cayman Chemical) in U-46619-constricted lungs from each group. Resta and Walker (33) have previously employed these concentrations of each NO donor to elicit vasodilation in isolated rat lungs. Parallel experiments were performed with 1 µM N-acetylpenicillamine (Sigma) and 2 µM spermine (Sigma) to test for possible nonspecific actions of SNAP and spermine NONOate, respectively.
Western Blotting for eNOS
To assess whether pulmonary eNOS levels are elevated by estrogen, separate sets of rats from each group were anesthetized with intraperitoneal pentobarbital sodium (25 mg) and their lungs were quickly snap-frozen in liquid nitrogen. Whole right lungs were fragmented with a mortar and pestle cooled in liquid nitrogen, then were homogenized on ice in 10 mM Tris · HCl buffer (pH 7.4) containing 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 4 µM pepstatin A, 1 µM aprotinin, and 2 mM phenylmethylsulfonyl fluoride (all from Sigma). Homogenates were centrifuged at 1,500 g at 4°C for 10 min to remove tissue debris. Protein concentrations of samples were determined by the Bradford method (Bio-Rad protein assay). Tissue sample proteins were resolved by SDS-PAGE with 7.5% acrylamide gels. In addition to the samples, each gel included both molecular mass (Bio-Rad) and eNOS (human endothelial lysate; Transduction Laboratories) standards. The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked overnight at 4°C with 5% nonfat milk, 3% BSA (Sigma), and 0.05% Tween 20 (Bio-Rad) in a Tris-buffered saline solution (TBS) containing 10 mM Tris · HCl and 50 mM NaCl (pH 7.5). Blots were incubated for 4 h at room temperature with a mouse monoclonal antibody raised against human eNOS (1:2,500; Transduction Laboratories) in TBS. Immunochemical labeling was achieved by incubation for 1 h at room temperature with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; Bio-Rad) in TBS followed by chemiluminescence labeling (Amersham ECL). eNOS protein bands were detected by exposure to chemiluminescence-sensitive film (Kodak). Membranes were stained with Coomassie brilliant blue to confirm equal protein loading per lane.Calculations and Statistics
Vascular morphometry. External and luminal arterial diameters were calculated from the medial and luminal circumferences, respectively. Arterial wall thickness was assessed by subtracting the luminal radius from the external radius and is expressed as a percent of external diameter according to the formula [(2 × wall thickness)/external diameter] × 100.
Isolated lung experiments.
Total pulmonary vascular resistance in isolated perfused lungs was
calculated as the difference between Pa and Pv
divided by flow (30 ml · min1 · kg body
wt
1). Pulmonary arterial resistance was calculated as the
difference between Pa and Pc divided by flow.
Similarly, pulmonary venous resistance was calculated as the difference
between Pc and Pv divided by flow. Vasodilatory
responses were calculated as percent reversal of U-46619-induced
vasoconstriction for the total pulmonary vasculature as well as for
arterial and venous segments.
Western blotting. eNOS protein bands from samples were quantitated by densitometric analysis (SigmaGel, SPSS) and normalized to those of the eNOS standard to allow statistical comparisons between blots.
All data are expressed as means ± SE. Values of n are the number of vessels in each group for statistical comparisons of vessel wall area; for all other comparisons, n is the number of animals in each group. Where appropriate, a one-way or two-way ANOVA was used to make comparisons. If differences were detected by ANOVA, individual groups were compared with the Student-Newman-Keuls test. A probability of P < 0.05 was accepted as significant for all comparisons. ![]() |
RESULTS |
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Plasma E2 and Uterine Weight
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Chronic Hypoxia-Induced Right Ventricular Hypertrophy, Polycythemia, and Arterial Remodeling
Greater RV-to-T ratios were observed for CH intact rats compared with normoxic control rats (Fig. 1), thus demonstrating right ventricular hypertrophy indicative of pulmonary hypertension. Furthermore, whereas ovariectomy exacerbated the development of right ventricular hypertrophy in vehicle-treated animals, E2
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CH intact rats exhibited polycythemia as evidenced by a significantly
greater hematocrit compared with normoxic intact animals (Fig.
2). This polycythemic response was
potentiated by ovariectomy in vehicle-treated rats but was
significantly lower in OVX rats receiving E2 relative to
both CH intact and CH OVX vehicle groups.
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Figure 3 illustrates percent wall
thickness for small pulmonary arteries with external diameters of
20-50 µm (Fig. 3A) and 51-100 µm (Fig.
3B) from each group of rats. Chronic hypoxia was associated
with increased wall thickness in all groups compared with normoxic
control vessels. Furthermore, wall thickness was greater in OVX
vehicle-treated animals compared with CH intact rats, and
E2 replacement largely attenuated arterial remodeling as
indicated by the smaller percent wall thickness compared with both CH
intact and CH OVX vehicle groups for each range of vessel diameters.
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Isolated Lung Experiments
Baseline segmental vascular resistances and responses to U-46619.
Table 2 shows total, arterial, and venous
baseline resistances in isolated perfused lungs from each group of
animals. Total and arterial baseline resistances were greater in all
hypoxic groups compared with normotensive control groups as previously demonstrated in lungs from male rats (29, 31-33).
Because the pulmonary circulation of CH rats exhibits no detectable
basal tone in this preparation (32), these data provide
functional evidence for chronic hypoxia-induced arterial remodeling.
Additionally, total and arterial baseline resistances tended to be
greater in lungs from CH OVX vehicle-treated rats compared with those
from other CH groups, although significance was achieved only for total resistance between CH OVX vehicle and CH intact groups. These data are
consistent with the more profound arterial remodeling observed in OVX
rats treated with vehicle (Fig. 3). L-NNA was without
effect on baseline resistances as Resta and colleagues (31,
33) have reported previously, suggesting that endogenous NO does
not contribute to the maintenance of low basal tone in this
preparation. There were no differences in baseline venous resistance
between groups.
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Segmental vasodilatory responses to ionomycin.
Lungs isolated from CH intact and CH OVX vehicle rats exhibited greater
total vasodilatory responses to the non-receptor-mediated, EDNO-dependent vasodilator ionomycin compared with normoxic control rats (Fig. 4A) as Resta and
colleagues (29, 33) have previously described for CH male
rats. Although arterial reactivity to ionomycin also tended to be
augmented after chronic hypoxia, significance was achieved only in the
CH OVX vehicle group. Contrary to our hypothesis, E2
replacement did not further enhance EDNO-dependent responses but rather
yielded total and arterial dilations that were not significantly
different from those of the normoxic intact group. Furthermore, lungs
isolated from CH OVX vehicle-treated rats showed a tendency for greater
arterial responsiveness to ionomycin versus that in CH intact and CH
OVX E2
groups, although a significant difference was
observed only versus the CH intact group. Venous responses were not
different between groups.
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Inhibition of responses to ionomycin by L-NNA.
The NOS inhibitor L-NNA attenuated total and arterial
reactivity to ionomycin in all groups (Fig. 4B), thus
demonstrating a contribution of EDNO to ionomycin-induced vasodilation.
Venous responsiveness was similarly inhibited by L-NNA,
with statistical differences noted in CH intact and CH OVX
E2 groups. However, this L-NNA-induced
attenuation was modest compared with that which Resta and Walker
(33) have previously observed in lungs from male rats.
Unexpectedly, lungs from CH OVX vehicle-treated rats exhibited greater
total and arterial vasodilation to ionomycin in the presence of
L-NNA compared with other groups.
Segmental vasodilatory responses to exogenous NO.
Total and segmental reactivity to the NO donors SNAP (Fig.
5A) and spermine NONOate (Fig.
5B) was attenuated in all CH groups compared with that in
normoxic control groups, suggesting that chronic hypoxia attenuates
pulmonary vascular smooth muscle sensitivity to exogenous NO. However,
no differences in reactivity to either SNAP or spermine NONOate were
observed between CH groups, indicating that NO-dependent
vasodilation is not further altered by either ovariectomy or
E2 replacement. N-acetylpenicillamine and
spermine exhibited no apparent vasoactive properties in lungs from
normoxic intact rats (data not shown; n = 2 for each
compound).
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eNOS Protein Levels
Immunoreactive eNOS was detected in lungs from all groups as a single band of ~140 kDa (Fig. 6). Greater quantities of eNOS were observed in lungs from CH versus normoxic control groups as previously reported for male rats (20, 29, 30, 35). Although eNOS levels tended to be greater in lungs from CH OVX vehicle-treated animals compared with both CH intact and CH OVX E2
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DISCUSSION |
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The present study examined effects of estrogen on CH pulmonary
hypertension and determined the ability of estrogen to regulate pulmonary eNOS expression and associated EDNO-dependent reactivity in
this setting. The major findings from this study are that 1) ovariectomy exacerbated the right ventricular hypertrophy,
polycythemia, and pulmonary arterial remodeling that result from
long-term hypoxic exposure and E2 replacement prevented
these responses to ovariectomy; 2) whereas chronic hypoxia
augmented pulmonary vasodilatory responsiveness to the EDNO-dependent
dilator ionomycin, E2
replacement did not potentiate
this response; 3) E2
was without effect on
pulmonary vasodilatory responses to exogenous NO, although NO-dependent reactivity was attenuated after chronic hypoxia; and 4)
chronic hypoxia-induced upregulation of pulmonary eNOS was unaltered by either ovariectomy or E2
replacement. These findings
suggest that estrogen exerts a protective influence in the hypertensive pulmonary circulation but that this protection is not likely a function
of increased eNOS expression.
The initial observation of a gender difference in the development of
chronic hypoxia-induced pulmonary hypertension was made by Burton et
al. (6), who noted that female chickens raised at 3,810 m
developed less pulmonary hypertension and right ventricular hypertrophy
than males. Subsequent studies (22, 28) have demonstrated a similar sexually dimorphic pattern in both swine and rats. Our present findings that ovariectomy augmented right ventricular hypertrophy, arterial remodeling, and polycythemic responses to long-term hypoxia agree with a previous report (26) of
increased hematocrit and right ventricle weight after ovariectomy in CH rats and support a role for ovarian hormones in mediating the different susceptibility to hypoxic pulmonary hypertension
between the sexes. Indeed, E2 replacement
prevented each of these responses to ovariectomy, suggesting that
decreased levels of circulating E2
account for the
greater pulmonary hypertension that follows ovariectomy.
Consistent with these data are previous studies demonstrating that
pharmacological doses of E2
administered to male rats
suppress right ventricular hypertrophy and arterial remodeling
associated with monocrotaline-induced pulmonary hypertension
(10) as well as polycythemia resulting from chronic
hypoxia (24). Chronic E2
treatment in utero
has further been shown to decrease pulmonary vascular resistance and
remodeling in perinatal pulmonary hypertension (27).
However, the mechanisms by which E2
attenuates pulmonary hypertension are not presently understood.
Considering the known stimulatory effect of E2 on
vascular eNOS expression (17, 18, 21), we hypothesized
that E2
-induced attenuation of hypoxic pulmonary
hypertension was associated with upregulation of pulmonary eNOS and
enhanced EDNO-dependent reactivity. Contrary to our hypothesis,
E2
-treatment in CH OVX rats neither increased pulmonary
eNOS levels nor augmented vasodilatory responsiveness to the
EDNO-mediated agonist ionomycin. In contrast, although reactivity to
ionomycin was greater in both CH intact and CH OVX vehicle-treated
groups compared with normoxic control groups, as Resta and colleagues
(29, 33) have previously observed after chronic hypoxia in
male rats, no such change in reactivity was observed in the CH OVX
E2
group. The reason for the apparently reduced
reactivity to ionomycin after administration of E2
is not clear. Previous studies from our laboratory (29, 33)
suggest that the upregulation of pulmonary eNOS associated with chronic hypoxia in male rats is dependent on altered vascular mechanical forces
associated with pulmonary hypertension as opposed to hypoxia per se.
Therefore, it is possible that the lesser pulmonary hypertension and
vascular remodeling observed in E2
-treated rats in the
present study limited the induction of pulmonary eNOS by chronic
hypoxia, which could potentially have masked any direct influence of
E2
on eNOS expression. The correlation between
accentuated right ventricular hypertrophy, arterial remodeling, and
reactivity to ionomycin after ovariectomy alone is consistent with this
possibility. Although it is additionally possible that any positive
influence of E2
on eNOS activity was complicated by
E2
-induced decreases in vascular smooth muscle
sensitivity to NO, the similar responses to the NO donors SNAP and
spermine NONOate observed between CH groups suggest that reactivity to
exogenous NO was unaltered by either ovariectomy or E2
treatment.
Although our present findings do not support a role for altered eNOS
expression in mediating the inhibitory effect of E2 on
hypoxic pulmonary hypertension, they also do not preclude the possibility that this response is dependent on changes in endothelial function. Accumulating evidence suggests that decreased synthesis of
the endothelium-derived vasoconstrictor and mitogenic factor endothelin-1 (ET-1) contributes to the antiatherosclerotic properties of E2
(2, 3, 39). ET-1 has additionally
been implicated in mediating the vascular remodeling component of
chronic hypoxia-induced pulmonary hypertension (5, 7) and
may facilitate the pulmonary vasoconstrictor response to acute hypoxia
via suppression of ATP-sensitive K+ channel activity
(34). Furthermore, ET-1 synthesis is elevated with chronic
exposure to hypoxia and appears to contribute to increased basal tone
in isolated lungs from CH rats after NO synthesis inhibition
(25). Therefore, the inhibitory effect of estrogen on the
development of HPV could potentially result from decreased ET-1
expression. Considering the apparent contribution of cyclooxygenase products to the inhibitory effects of E2
on HPV in
isolated ovine lungs (15), an additional possibility is
that the protective influence of estrogen is a consequence of increased
prostacyclin production. It is also possible that basal production of
NO is increased secondary to the acute stimulation of pulmonary eNOS by
E2
(19). Alternatively, the lesser right
ventricular hypertrophy and arterial remodeling observed in
E2
-treated animals may result from direct vasodilatory
(12, 16) and antimitogenic effects of E2
on
pulmonary vascular smooth muscle (9, 37) or, rather, to
the upregulation of inducible NOS (iNOS) by E2
as
previously reported in systemic vascular tissue (4).
However, this latter possibility is unlikely considering that we
observed no differences in pulmonary iNOS expression between groups as
assessed by Western blotting (data not shown), whereas large increases
in iNOS levels are apparent in lungs from rats treated with
lipopolysaccharide (31).
It is noteworthy that the inhibitory effects of L-NNA on
ionomycin-induced pulmonary vasodilation were rather modest compared with previously published observations from male rats
(33). Whether the residual dilation to ionomycin in the
presence of L-NNA is a result of incomplete NOS inhibition
is not clear. This possibility seems unlikely, however, considering
that a previous study from our laboratory (33) has
demonstrated that the 300 µM dose of L-NNA currently
employed nearly abolishes arterial dilation to the EDNO-dependent
pulmonary vasodilators arginine, vasopressin, and ET-1 in this
preparation. Furthermore, preliminary studies indicate that this
dose of L-NNA largely inhibits the accumulation of nitrite
and nitrate in the perfusate of isolated saline-perfused rat lungs
(Walker BR, Resta TC, and Nelin LD, unpublished observation),
suggesting effective inhibition of NO synthesis. Alternatively, it is
possible that an additional endothelium-dependent vasodilator
contributes to ionomycin-induced vasodilation in this preparation.
Because meclofenamate was added to the perfusate to inhibit
prostaglandin synthesis, involvement of an endothelium-derived hyperpolarizing factor in the response to ionomycin is a likely possibility. A further unexpected finding was the greater total and
arterial dilation to ionomycin in lungs from CH OVX vehicle-treated animals compared with other groups after NOS inhibition. Whether this
greater reactivity after ovariectomy represented decreased responsiveness to L-NNA is not clear because the unpaired
nature of the experiments precluded statistical comparisons of percent inhibition by L-NNA. An additional complicating factor is
the greater constriction to U-46619 observed in
L-NNA-treated versus untreated lungs in the CH OVX group.
Finally, it is possible that the decreased endogenous synthesis of
E2 associated with ovariectomy in vehicle-treated
animals resulted in a compensatory increase in an alternative
endothelium-dependent vasodilator. Nevertheless, the inhibitory effect
of L-NNA on ionomycin-induced pulmonary vasodilation
demonstrated a significant contribution of endogenous NO to this
response, and the responses to ionomycin correlated in magnitude with
pulmonary vascular eNOS levels, neither of which are potentiated by
E2
.
An interesting observation from these studies is the reduced reactivity
observed to both SNAP and spermine NONOate in lungs isolated from CH
rats compared with those from normoxic control rats. These findings
suggest that pulmonary vascular smooth muscle sensitivity to NO is
reduced after exposure to chronic hypoxia, which is at odds with
previously published observations (33) from our laboratory
demonstrating no effect of chronic hypoxia on vasodilatory
responsiveness to the same NO donors in lungs from male rats. The
reason for this discrepancy is not presently clear but could represent
a gender difference independent of E2.
In conclusion, we have demonstrated that E2 mediates an
inhibitory influence on chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia that is
not associated with upregulation of pulmonary eNOS or enhanced EDNO-dependent responsiveness. Further investigation is required to
determine whether this protective effect of E2
is
attributable to decreased pulmonary ET-1 expression, to acute
stimulation of eNOS activity, or to direct vasodilatory and
antimitogenic properties of E2
in vascular smooth muscle.
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ACKNOWLEDGEMENTS |
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We thank Minerva Murphy, Dr. Rayna Gonzales, Melissa Montaño, and Ann Ommani for expert technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-09660 (T. C. Resta), HL-03852 (N. L. Kanagy), and HL-58124 (B. R. Walker); by a Scientist Development Grant from the American Heart Association (T. C. Resta); and by dedicated health research funds of the University of New Mexico School of Medicine (T. C. Resta).
T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: T. C. Resta, 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: tresta{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 6 April 2000; accepted in final form 27 July 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akishita, M,
Kozaki K,
Saito T,
Eto M,
Ishikawa M,
Miyoshi H,
Hashimoto M,
Toba K,
Orimo H,
and
Ouchi Y.
Effects of estrogen on atherosclerosis formation and serum nitrite/nitrate concentrations in cholesterol-fed ovariectomized rabbits.
J Atheroscler Thromb
3:
114-119,
1996[Medline].
2.
Akishita, M,
Ouchi Y,
Miyoshi H,
Orimo A,
Kozaki K,
Eto M,
Ishikawa M,
Kim S,
Toba K,
and
Orimo H.
Estrogen inhibits endothelin-1 production and c-fos gene expression in rat aorta.
Atherosclerosis
125:
27-38,
1996[ISI][Medline].
3.
Barton, M,
Haudenschild CC,
d'Uscio LV,
Shaw S,
Munter K,
and
Luscher TF.
Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice.
Proc Natl Acad Sci USA
95:
14367-14372,
1998
4.
Binko, J,
and
Majewski H.
17-Estradiol reduces vasoconstriction in endothelium-denuded rat aortas through inducible NOS.
Am J Physiol Heart Circ Physiol
274:
H853-H859,
1998
5.
Bonvallet, ST,
Zamora MR,
Hasunuma K,
Sato K,
Hanasato N,
Anderson D,
Sata K,
and
Stelzner TJ.
BQ123, an ETA-receptor antagonist, attenuates hypoxic pulmonary hypertension in rats.
Am J Physiol Heart Circ Physiol
266:
H1327-H1331,
1994
6.
Burton, RR,
Besch EL,
and
Smith AH.
Effect of chronic hypoxia on the pulmonary arterial blood pressure of the chicken.
Am J Physiol
214:
1438-1442,
1968[ISI][Medline].
7.
DiCarlo, VS,
Chen SJ,
Meng QC,
Durand J,
Yano M,
Chen YF,
and
Oparil S.
ETA-receptor antagonist prevents and reverses chronic hypoxia-induced pulmonary hypertension in rat.
Am J Physiol Lung Cell Mol Physiol
269:
L690-L697,
1995
8.
Dupon, C,
and
Kim MH.
Peripheral plasma levels of testosterone, androstenedione, and oestradiol during the rat oestrous cycle.
J Endocrinol
59:
653-654,
1973[ISI][Medline].
9.
Espinosa, E,
Oemar BS,
and
Luscher TF.
17 beta-Estradiol and smooth muscle cell proliferation in aortic cells of male and female rats.
Biochem Biophys Res Commun
221:
8-14,
1996[ISI][Medline].
10.
Farhat, MY,
Chen MF,
Bhatti T,
Iqbal A,
Cathapermal S,
and
Ramwell PW.
Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats.
Br J Pharmacol
110:
719-723,
1993[Abstract].
11.
Feddersen, CO,
Chang S,
Czartalomna J,
and
Voelkel NF.
Arachidonic acid causes cyclooxygenase-dependent and -independent pulmonary vasodilation.
J Appl Physiol
68:
1799-1808,
1990
12.
Gonzales, RJ,
and
Kanagy NL.
Endothelium-independent relaxation of vascular smooth muscle by 17-estradiol.
J Cardiovasc Pharmacol
4:
227-234,
1999.
13.
Gonzales, RJ,
and
Kanagy NL.
Regulation of eNOS expression by 17-estradiol in the lung and thoracic aorta (Abstract).
FASEB J
14:
A404,
2000.
14.
Gonzales, RJ,
Walker BR,
and
Kanagy NL.
17-Estradiol increases endothelium-dependent vasodilation in pulmonary but not peripheral arteries (Abstract).
FASEB J
12:
A491,
1998[ISI].
15.
Gordon, JB,
Wetzel RC,
McGeady ML,
Adkinson NF, Jr,
and
Sylvester JT.
Effects of indomethacin on estradiol-induced attenuation of hypoxic vasoconstriction in lamb lungs.
J Appl Physiol
61:
2116-2121,
1986
16.
Jiang, CW,
Sarrel PM,
Lindsay DC,
Poole-Wilson PA,
and
Collins P.
Endothelium-independent relaxation of rabbit coronary artery by 17 beta-oestradiol in vitro.
Br J Pharmacol
104:
1033-1037,
1991[Abstract].
17.
Kauser, K,
and
Rubanyi GM.
Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.
J Vasc Res
34:
229-236,
1997[ISI][Medline].
18.
Kleinert, H,
Wallerath T,
Euchenhofer C,
Ihrig-Biedert I,
Li H,
and
Förstermann U.
Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved.
Hypertension
31:
582-588,
1998
19.
Lantin-Hermoso, RL,
Rosenfeld CR,
Yuhanna IS,
German Z,
Chen Z,
and
Shaul PW.
Estrogen acutely stimulates nitric oxide synthase activity in fetal pulmonary artery endothelium.
Am J Physiol Lung Cell Mol Physiol
273:
L119-L126,
1997
20.
Le Cras, TD,
Xue C,
Rengasamy A,
and
Johns RA.
Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung.
Am J Physiol Lung Cell Mol Physiol
270:
L164-L170,
1996
21.
MacRitchie, AN,
Jun SS,
Chen Z,
German Z,
Yuhanna IS,
Sherman TS,
and
Shaul PW.
Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium.
Circ Res
81:
355-362,
1997
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.
Meyrick, B,
and
Reid L.
Ultrastructural features of the distended pulmonary arteries of the normal rat.
Anat Rec
193:
71-98,
1979[ISI][Medline].
24.
Moore, LG,
McMurtry IF,
and
Reeves JT.
Effects of sex hormones on cardiovascular and hematologic responses to chronic hypoxia in rats.
Proc Soc Exp Biol Med
158:
658-662,
1978.
25.
Muramatsu, M,
Rodman DM,
Oka M,
and
McMurtry IF.
Endothelin-1 mediates nitro-L-arginine vasoconstriction of hypertensive rat lungs.
Am J Physiol Lung Cell Mol Physiol
272:
L807-L812,
1997
26.
Ou, LC,
Sardella GL,
Leiter JC,
Brinck-Johnsen T,
and
Smith RP.
Role of sex hormones in development of chronic mountain sickness in rats.
J Appl Physiol
77:
427-433,
1994
27.
Parker, TA,
Ivy DD,
Galan HL,
Grover TR,
Kinsella JP,
and
Abman SH.
Estradiol improves pulmonary hemodynamics and vascular remodeling in perinatal pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
278:
L374-L381,
2000
28.
Rabinovitch, M,
Gamble WJ,
Miettinen OS,
and
Reid L.
Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery.
Am J Physiol Heart Circ Physiol
240:
H62-H72,
1981
29.
Resta, TC,
Chicoine LG,
Omdahl JL,
and
Walker BR.
Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia.
Am J Physiol Heart Circ Physiol
276:
H699-H708,
1999
30.
Resta, TC,
Gonzales RJ,
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
31.
Resta, TC,
O'Donaughy TL,
Earley S,
Chicoine LG,
and
Walker BR.
Unaltered vasoconstrictor responsiveness after iNOS inhibition in lungs from chronically hypoxic rats.
Am J Physiol Lung Cell Mol Physiol
276:
L122-L130,
1999
32.
Resta, TC,
Sanders TC,
Eichinger MR,
Crowley MR,
and
Walker BR.
Segmental vasodilatory effectiveness of inhaled NO in lungs from chronically hypoxic rats.
Respir Physiol
114:
161-173,
1998[ISI][Medline].
33.
Resta, TC,
and
Walker BR.
Chronic hypoxia selectively augments endothelium-dependent pulmonary arterial vasodilation.
Am J Physiol Heart Circ Physiol
270:
H888-H896,
1996
34.
Sato, K,
Morio Y,
Morris KG,
Rodman DM,
and
McMurtry IF.
Mechanism of hypoxic pulmonary vasoconstriction involves ETA receptor-mediated inhibition of KATP channel.
Am J Physiol Lung Cell Mol Physiol
278:
L434-L442,
2000
35.
Shaul, PW,
North AJ,
Brannon TS,
Ujiie K,
Wells LB,
Nisen PA,
Lowenstein CJ,
Snyder SH,
and
Star RA.
Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.
Am J Respir Cell Mol Biol
13:
167-174,
1995[Abstract].
36.
Sunyer, J,
Anto JM,
McFarlane D,
Domingo A,
Tobias A,
Barcelo MA,
and
Munoz A.
Sex differences in mortality of people who visited emergency rooms for asthma and chronic obstructive pulmonary disease.
Am J Respir Crit Care Med
158:
851-856,
1998
37.
Suzuki, A,
Mizuno K,
Ino Y,
Okada M,
Kikkawa F,
Mizutani S,
and
Tomoda Y.
Effects of 17-estradiol and progesterone on growth-factor-induced proliferation and migration in human female aortic smooth muscle cells in vitro.
Cardiovasc Res
32:
516-523,
1996[ISI][Medline].
38.
Wetzel, RC,
Zacur HA,
and
Sylvester JT.
Effect of puberty and estradiol on hypoxic vasomotor response in isolated sheep lungs.
J Appl Physiol
56:
1199-1203,
1984
39.
Ylikorkala, O,
Orpana A,
Puolakka J,
Pyorala T,
and
Viinikka L.
Postmenopausal hormonal replacement decreases plasma levels of endothelin-1.
J Clin Endocrinol Metab
80:
3384-3387,
1995[Abstract].