17beta -Estradiol increases nitric oxide-dependent dilation in rat pulmonary arteries and thoracic aorta

Rayna J. Gonzales, Benjimen R. Walker, and Nancy L. Kanagy

Vascular Physiology Group, Department of Cell Biology and Physiology, Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131-5218


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Past studies have demonstrated that 17beta -estradiol (E2beta ) 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 E2beta regulation of NO synthesis in the pulmonary vasculature. The present study evaluated E2beta regulation of eNOS function in pulmonary arteries and thoracic aortas. We hypothesized that E2beta 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 E2beta 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 E2beta -treated group was enhanced compared with that in control animals. Endothelium-intact aortic rings from E2beta -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 E2beta -treated compared with control rats. However, mRNA levels did not differ between groups. Thus we conclude that in vivo E2beta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -estradiol (E2beta ) 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 E2beta 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 E2beta 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 E2beta had increased expression of eNOS that was dose dependent (16). In addition, E2beta 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 E2beta 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 E2beta augments vasoconstriction in pulmonary arteries, so the effect of E2beta 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, E2beta 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 E2beta regulation of NO synthesis and release in other vascular beds when E2beta is maintained at physiological rather than pharmacological levels. Therefore, these experiments examined eNOS expression and activity in the systemic and pulmonary vasculatures after E2beta replacement therapy in ovariectomized (OVX) female rats. We hypothesized that physiological levels of E2beta would upregulate eNOS expression, leading to enhanced synthesis and release of NO in both the systemic and pulmonary vasculatures.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 E2beta stores, mini-osmotic pumps (ALZET model 2002) containing lipid-soluble E2beta (20 µg/24 h; Sigma) or vehicle (polypropylene glycol) were implanted subcutaneously at the base of the neck.

Isolated Aortic Ring Tissue Bath Preparation

After 7 days of treatment, E2beta - and vehicle-treated animals were anesthetized with pentobarbital sodium (50 mg/kg), heparinized, and exsanguinated. Thoracic aortas were removed and placed in ice-cold physiological salt solution (PSS) containing (in mmol/l) 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 · 7H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2-EDTA, and 1.6 CaCl2. Aortas were cleaned of adventitia and cut into 4-mm rings, and when necessary, endothelium was removed by gently rubbing the lumen with fine-tipped forceps. Next, tissues were suspended in water-jacketed tissue baths filled with PSS maintained at 37°C and aerated with 95% O2 and 5% CO2. Rings were stretched with 2.5 g of passive tension to allow maximal detection of active tension generation as determined in preliminary studies and were equilibrated for 1 h. After a 30-min incubation with indomethacin (10 µmol/l; cyclooxygenase inhibitor), rings were challenged with phenylephrine (PE; 10-6 mol/l), and the presence of endothelium was verified by ACh (10-6 mol/l) relaxation. Tissues exhibiting <80% relaxation were omitted from the study. Force was continuously recorded with an FT03 transducer (Grass) and chart recorder (Gould RS 3800).

Isolated Aortic Ring Tissue Bath Protocol

Contractility studies determine if E2beta treatment in vivo enhances endothelium-dependent relaxation. To determine whether chronic in vivo E2beta 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 Nomega -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 E2beta -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 E2beta or vehicle for 7 days to determine whether E2beta upregulates eNOS activity in the pulmonary vasculature. Each rat was anesthetized with pentobarbital sodium, and the trachea was cannulated to ventilate the lungs at a frequency of 55 breaths/min and a tidal volume of 2.5 ml with warmed and humidified gas (6% CO2, 21% O2, and balance N2). Inspiratory pressure was set at 9 cmH2O and positive end-expiratory pressure at 2 cmH2O. Heparinized whole blood was withdrawn from the heart to measure plasma E2beta levels with a radioimmunoassay (DiSorin). The pulmonary artery was cannulated with a 13-gauge needle stub and perfused with PSS containing 4% albumin and 10 µg/ml of meclofenamate (cyclooxygenase inhibitor). The left ventricle was cannulated, and the heart and lungs were removed en bloc and suspended in a humidified chamber maintained at 37°C. Initial perfusion was set at 0.9 ml/min and then was gradually increased to 30 ml · min-1 · kg body wt-1 and maintained at this rate for the duration of the experiment. Lungs were equilibrated for 30 min to allow pulmonary arterial pressure (Pa) to reach steady state. During the experiment, lungs were perfused in zone three conditions [Pa > pulmonary venous pressure (Pv) > airway pressure (Paw)], with venous pressure ~3 Torr. Pa and Pv were recorded with pressure transducers (model P23XL, Spectro) and a chart recorder (model 3400, Gould). Data were stored and processed with a computer-based data analysis and acquisition system (CODAS, Dataq Instruments).

Isolated 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 E2beta increased eNOS activity in pulmonary arteries, we examined total, arterial, and venous vasodilatory responses to eNOS stimulation by ionomycin (calcium ionophore) in lungs from E2beta - 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.

Double occlusion was performed by simultaneously occluding the arterial inflow and venous outflow for <5 s. Vascular pressures equilibrated to microvascular perfusion pressure to yield an assessment of Pcap. According to previous studies (6, 36), this method of vascular occlusion yields accurate estimates of Pcap. In a separate set of experiments, the NO contribution to dilation was assessed by pretreating isolated lungs with L-NNA (300 µmol/l) using the same protocol described in Isolated Perfused Lung Protocol. This dose of L-NNA is effective in inhibiting endothelium-derived NO-dependent pulmonary vasodilation in the isolated perfused rat lung (27).

Western Blot Analysis

eNOS expression in aortas and lung homogenates from E2beta - and vehicle-treated rats. eNOS protein content was evaluated with standard immunoblotting methods. The left lobes of the lungs and the thoracic aortas from E2beta - 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 E2beta 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 E2beta - and vehicle-treated rats. To further establish if E2beta 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 E2beta - 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 E2beta - 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 E2beta - 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.

                              
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Table 1.   Accession nos., expected PCR product sizes of each fragment product length, and primer sequences for eNOS and MDH

eNOS mRNA expression in thoracic aortas and lungs from E2beta - 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 at -80°C. For the isolated lung study, L-NNA was dissolved in PSS perfusate before the experiments. Fixative solutions were prepared by dissolving 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffer (0.1 mol/l), and ddH2O. Phosphate buffer (0.2 mol/l) containing Na2HPO4 (dibasic; 0.05 mol/l) and NaCl (0.14 mol/l) was diluted in ddH2O, and the pH was adjusted to 7.4 with concentrated HCl. Homogenizing buffer containing sucrose (225 mmol/l), Tris · HCl (10 mmol/l, pH 7.4), and EDTA (2 mmol/l) diluted in ddH2O was prepared on the same day the tissues were homogenized. Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were diluted in ddH2O and stored as stock solutions at -20°C.

Statistics

Data are reported as means ± SE, with P <=  0.05 considered significant. Groups were compared with Student's t-test of significance. In the contractility studies, individual points on concentration-response curves for the two groups were compared with Student's t-test. Data expressed as percentages were normalized with the arcsine transformation before statistical analysis. The n value is the number of animals used for each study.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma E2beta Concentrations in OVX Female Rats

E2beta plasma levels were significantly higher in rats treated with E2beta (102 ± 11 pg/ml) compared with those treated with vehicle (<5 pg/ml). E2beta replacement resulted in plasma levels in the physiological range (1, 3, 15, 18, 30), demonstrating that E2beta replacement via osmotic pumps delivered a dose of E2beta in OVX rats that mimicked endogenous levels.

Effects of E2beta In Vivo on Endothelium-Dependent Relaxation Induced by ACh in Aortic Rings

Vasodilatory responses to ACh in endothelium-intact aortic rings from OVX rats treated with E2beta or vehicle are summarized in Fig. 1A. Cumulative concentration-response curves to ACh (10-10 to 10-4.5 mol/l) were generated in tissues contracted with PE (10-6 mol/l). Force generated by PE was not different between groups (Fig. 1A, inset). Relaxation to the 10-7 and 10-6.5 mol/l doses of ACh was significantly enhanced in endothelium-intact aortic rings from E2beta -treated OVX rats. NOS inhibition by L-NNA (100 µmol/l) attenuated ACh-induced relaxation, indicating that NO generation leads to the augmented response to ACh in aortas from E2beta -treated rats (data not shown).


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Fig. 1.   Contractility data from aortic rings isolated from ovariectomized (OVX) vehicle (Veh)- and 17beta -estradiol (E2beta )-treated rats (n = 5/group). A: cumulative concentration-response curves to ACh (10-10 to 10-4.5 mol/l) were generated in endothelium-intact rings contracted with phenylephrine (PE; 10-6 mol/l). Data are means ± SE. There was no difference in PE contraction between groups (inset). However, relaxation to ACh was augmented in aortic rings from E2beta -treated rats. *P < 0.05 vs. control by Student's t-test. B: cumulative concentration-response curves to S-nitroso-N-penicillamine (SNAP) in denuded aortic rings from vehicle- and E2beta -treated rats (n = 4/group). Data are means ± SE. Relaxation was not different between groups.

Because E2beta 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 E2beta -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 E2beta -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 E2beta treatment of OVX rats.

Effects of E2beta Administration In Vivo on Endothelium-Dependent Dilation in Perfused Isolated Lungs

Table 2 illustrates total and segmental (arterial and venous) resistances before and after U-46619 constriction in lungs from vehicle- and E2beta -treated rats. There was no difference in baseline resistance between lungs from vehicle- and E2beta -treated rats. To elicit an increase in pulmonary vascular pressure (10 Torr), lungs were constricted with U-46619 (605 ± 35 nmol/l for vehicle and 534 ± 21 nmol/l for E2beta ). There were no differences in segmental resistances between groups after U-46619 administration. After the pressor response to U-46619 became stable, vasodilatory responses to ionomycin (1 µmol/l) were assessed (Fig. 2A). Total and arterial but not venous pulmonary vasodilatory responses to ionomycin were significantly greater in lungs from E2beta -treated compared with those from vehicle-treated animals.

                              
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Table 2.   Total and segmental pulmonary vascular resistances pre- and post-U-46619 constriction



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Fig. 2.   Total, arterial, and venous pulmonary vasodilatory responses to ionomycin (1 µmol/l) in isolated lungs from vehicle- and E2beta -treated rats. A: E2beta treatment augmented arterial vasodilatory responses to ionomycin in lungs from OVX rats compared with those in vehicle-treated rats (n = 8/group). *P < 0.05 vs. vehicle. B: Nomega -nitro-L-arginine (L-NNA; 300 µmol/l) abolished the differences in ionomycin vasodilation between lungs from E2beta - and vehicle-treated rats (n = 5/group). Data are means ± SE. Arcsine transformed data were compared with Student's t-test.

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 E2beta -treated rats was NO mediated, lungs were treated with the nonselective NOS inhibitor L-NNA (300 µmol/l). Table 3 shows total, arterial, and venous baseline resistance and U-46619-constricted resistance in lungs from vehicle- and E2beta -treated rats in the presence of L-NNA. There were no differences in baseline resistances between groups. U-46619 elicited an increase in total, arterial, and venous vascular resistances from baseline resistance. Additionally, the change in vascular resistances induced by U-46619 was similar in lungs from both groups. However, a smaller dose of U-46619 was needed to achieve the same 10 Torr constriction in lungs with L-NNA compared with L-NNA vehicle-treated lungs (299 ± 24 vs. 605 ± 35 nmol/l for vehicle and 276 ± 9 vs. 534 ± 21 nmol/l for E2beta under L-NNA and L-NNA vehicle conditions, respectively). After the U-46619 constriction stabilized, vasodilatory responses to ionomycin (1 µmol/l) were assessed. L-NNA treatment annulled the differences in ionomycin-induced vasodilation in lungs from E2beta -treated compared with vehicle-treated rats (Fig. 2B). However, the vasodilatory response to ionomycin was not completely blocked by L-NNA in either group of lungs, suggesting that a vasodilatory component other than NO is involved in ionomycin-induced pulmonary vasodilation.

                              
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Table 3.   Total and segmental pulmonary vascular resistances pre- and post-U-46619 constriction in the presence of NOS inhibition with L-NNA

Western Blot Analysis of Endothelial Expression of eNOS in Aortas and Lungs From E2beta - and Vehicle-Treated OVX Rats

To determine if enhanced endothelium-dependent relaxation observed in aortas isolated from E2beta -treated rats was due to increased eNOS expression, eNOS levels were assessed in tissue homogenates from E2beta - and vehicle-treated rats. Figure 3A illustrates eNOS protein expression in aortic homogenates. Consistent with the contractility studies, there was a significant increase in eNOS protein expression in aortic tissue from E2beta -treated rats. This suggests that E2beta treatment in vivo for 7 days augments aortic endothelium-dependent relaxation by increasing NO production via elevated eNOS enzyme concentrations.


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Fig. 3.   Representative Western blots of endothelial nitric oxide synthase (eNOS) protein expression in tissue homogenates from vehicle- and E2beta -treated rats. A: eNOS expression was greater in aortic tissue homogenates isolated from E2beta -treated than that in homogenates from vehicle-treated animals (n = 4/group). B: eNOS expression in lung homogenates was not different between groups (n = 3 rats/group). Values are means ± SE of mean densitometry measurements of eNOS expression. *Significant differences between groups, P < 0.05 by Student's t-test.

Because lungs from E2beta -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 E2beta - and Vehicle-Treated OVX Rats

Figure 4A illustrates eNOS immunoreactivity in aortas from vehicle- and E2beta -treated animals. Consistent with the data from Western analysis, quantitative immunohistochemistry demonstrated a greater intensity of eNOS staining in aortas from E2beta -treated rats compared with aortas isolated from vehicle-treated rats. Moreover, the staining was localized within the endothelium. Photomicrographs of pulmonary arteries (~300 µm in diameter) stained with the eNOS antibody from vehicle- and E2beta -treated rats are illustrated in Fig. 4B. eNOS staining was significantly greater in pulmonary arteries from E2beta -treated compared with vehicle-treated rats. This suggests that the upregulation of eNOS occurs within the vasculature but is not detectable by Western analysis of whole lung homogenates.


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Fig. 4.   Quantitative immunohistochemical analysis of eNOS staining in tissues from vehicle- and E2beta -treated animals. A: eNOS staining in aortic sections was significantly greater in aortas from E2beta -treated rats (n = 7) and localized exclusively in the endothelium. B: eNOS-immunoreactive staining was also more intense in the endothelium of pulmonary arteries from E2beta -treated animals compared with arteries from vehicle-treated rats (n = 6). Data are means ± SE. *P < 0.05 vs. vehicle by Student's t-test.

Analysis of eNOS mRNA Levels in Aortas and Lungs From E2beta - and Vehicle-Treated OVX Rats

Because data from the aorta suggest that eNOS protein expression is increased in animals treated with E2beta , gene expression for eNOS was evaluated in the same tissue harvested for eNOS protein analysis. Figure 5A illustrates RPA data showing eNOS mRNA normalized to MDH mRNA in aortas from vehicle- and E2beta -treated OVX rats. There was no significant difference between groups. It appears that the E2beta -induced enhanced aortic vasodilation did not require sustained elevation of eNOS transcription. mRNA for eNOS was also assessed in total RNA isolated from lung tissue. Figure 5B illustrates eNOS mRNA normalized to MDH mRNA in lungs from vehicle- and E2beta -treated female rats. Similar to those observed in aortic tissue, eNOS mRNA levels were not different between groups.


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Fig. 5.   eNOS mRNA levels in tissues isolated from vehicle- and E2beta -treated female rats. MDH, malate dehydrogenase. A: aortic eNOS mRNA was not different between groups (n = 7 rats/group). B: eNOS mRNA levels in lung tissue isolated from vehicle-treated (n = 6) and E2beta -treated (n = 10) rats also did not differ. Data were normalized to MDH mRNA and are expressed as a percentage. Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

It has been suggested that estrogen directly modulates vascular tone by enhancing endothelium-dependent vasodilation. We observed that physiological levels of E2beta replacement in OVX rats augmented endothelium-dependent vasodilation and enhanced eNOS activity in aortas and pulmonary blood vessels. The observation that E2beta 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 E2beta treatment. These results expand the role for E2beta in protecting cardiovascular function.

The mechanisms by which E2beta increases endothelium-dependent relaxation have not been clearly elucidated. Some studies suggest that E2beta in vivo selectively enhances NO-dependent relaxation. For example, Cheng et al. (3) observed that aortic rings from rats chronically treated with E2beta 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 E2beta enhances endothelium-dependent relaxation of blood vessels by upregulating eNOS. Our direct measures of eNOS protein provide additional evidence for eNOS upregulation by E2beta .

Reports of estrogen actions in other vascular beds have demonstrated that E2beta 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 E2beta treatment on vasoreactivity and eNOS expression in both the aorta and the pulmonary vasculature. We hypothesized that if E2beta upregulates eNOS expression, then endothelium-dependent vasodilators would elicit greater NO-dependent relaxation in arteries from E2beta -treated animals.

In support of this hypothesis, we found that lungs isolated from E2beta -treated rats exhibited augmented vasodilatory responses to ionomycin and that aortas from E2beta -treated rats had augmented endothelium-dependent relaxation. Because E2beta 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, E2beta 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 E2beta during hypoxia, it did study the mechanisms associated with E2beta 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 E2beta -treated animals and that this treatment abolished the enhanced vasodilation in lungs from E2beta -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 E2beta enhances pulmonary endothelial cell NOS activity.

In aortic studies, the augmented component of the ACh-induced aortic relaxation by E2beta was also completely abolished by NOS inhibition. This is in agreement with the observation (19) that E2beta 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 E2beta treatment requires NOS.

Interestingly, Chang et al. (2) demonstrated that E2beta may stimulate prostacyclin synthesis in VSM. Like NO, prostacyclin is a potent pulmonary vasodilator capable of contributing to the enhanced vasodilatory effects that follow E2beta 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 E2beta - 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, E2beta may further enhance vasodilation by altering VSM sensitivity to NO. In postmenopausal women, short-term administration of E2beta augments vasodilatory responses to ACh in the coronary (11, 25) and systemic (35) circulations. These studies showed that E2beta improved both endothelium-dependent (ACh) and -independent (sodium nitroprusside) vasodilatory responses in coronary and forearm blood vessels, suggesting that E2beta 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 E2beta - and vehicle-treated rats. In addition, pulmonary vasodilation to NO donors was not augmented by E2beta treatment (Resta TC and Walker BR, unpublished data). Therefore, our data cannot be explained by enhanced VSM sensitivity to NO.

Many substances, including E2beta , 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 E2beta upregulation of NOS activity is less clear. Classically, the regulation of transcription by E2beta 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 E2beta upregulation of eNOS (16), an electromobility shift study (21) found that the estrogen receptor does not bind to the NOS promoter. Therefore, E2beta regulation of eNOS expression remains controversial. With Western analysis, we found that E2beta 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 E2beta -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 E2beta .

To date, no studies have investigated the effect of E2beta treatment in vivo on NOS expression in the pulmonary vasculature. However, an in vitro study (20) has demonstrated that chronic E2beta administration upregulates eNOS gene expression in fetal pulmonary arterial endothelial cells. Thus this study suggests that E2beta may augment pulmonary vasodilation by upregulating the NO-generating enzyme eNOS.

We found that E2beta 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 E2beta -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 E2beta - and vehicle-treated groups with this method. One possibility for the disparate findings with these two techniques is that the level of E2beta 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 E2beta -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 E2beta . It is interesting that other studies (14, 32) reporting increased eNOS protein levels after E2beta 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 E2beta treatment are due to elevated NOS expression but not to persistent increases in eNOS message.

In conclusion, replacing E2beta 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 E2beta -treated female rats is due to increased levels of the NO-generating enzyme eNOS. Enhanced eNOS levels may thus contribute to E2beta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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