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(Circulation Research. 1997;81:885-892.)
© 1997 American Heart Association, Inc.

Articles

17ß-Estradiol Regulation of Human Endothelial Cell Basal Nitric Oxide Release, Independent of Cytosolic Ca²⁺ Mobilization

Teresa Caulin-Glaser, G. García-Cardeña, Phillip Sarrel, William C. Sessa, , Jeffrey R. Bender

From the Division of Cardiovascular Medicine and the Raymond and Beverly Sackler Foundation Laboratory (T.C.-G., J.R.B.), the Department of Pharmacology (G.G.-C., W.C.S.), the Departments of Obstetrics and Gynecology and Psychiatry (P.S.), and the Molecular Cardiobiology Program, Boyer Center for Molecular Medicine (T.C.-G., G.G.-C., W.C.S., J.R.B.), Yale University School of Medicine, New Haven, Conn.

Correspondence to Jeffrey R. Bender, MD, Yale University School Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave, Room 454, New Haven, CT 06536-0812. E-mail jeffrey_bender{at}quickmail.yale.edu.

	Abstract

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Abstract Estradiol retards the development of atherosclerosis. Animal models have suggested that NO may be a critical effector molecule in this cardiovascular protection. In this study, female human umbilical vein endothelial cells (HUVECs) were propagated in phenol red–free gonadal hormone–free medium and pretreated with 17ß-estradiol (E₂). Reduced NO₂^- and NO₃^- (NO_X) concentration, determined by chemiluminescence, demonstrated a rapid increase in basal HUVEC NO release in response to physiological concentrations of E₂. The estrogen receptor (ER) antagonist ICI 164,384 inhibited the augmented NO release, demonstrating an ER-mediated component of this response. Because endothelial NO synthase (eNOS) activity is largely regulated by cytosolic Ca²⁺, relative [Ca²⁺]_i in response to E₂ was determined in a fluorometric assay. E₂ did not promote HUVEC Ca²⁺ fluxes. Furthermore, eNOS activity in E₂-pretreated endothelial whole-cell lysates was not dependent on additional Ca²⁺. Despite involving the ER, this is a nongenomic effect of E₂, as demonstrated by maintained responses in transcriptionally inhibited cells and by the rapidity (10 minutes) of cGMP formation in an NO bioassay. We demonstrate, for the first time, that independent of cytosolic Ca²⁺ mobilization, there is augmentation of eNOS activity with a resultant increase in HUVEC basal NO release in response to short-term estradiol exposure. Implications for the cardiovascular protective role of estrogen are discussed.

Key Words: atherosclerosis • estrogen • vascular endothelium • nitric oxide • nitric oxide synthase

	Introduction

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Estrogens protect against the development of coronary heart disease in women. Although they promote a more favorable lipid profile, their beneficial effect cannot be fully explained on this basis.^{1 2} In primate models, acute administration of E₂ rapidly restores endothelium-dependent dilation to atherosclerotic arteries, suggesting a direct effect of E₂ on vascular reactivity.³ The mechanism of this effect is not yet understood but may include rapid nongenomic effects on vascular tone as well as inhibitory and/or stimulatory effects of the hormone on gene transcription and cell proliferation.

NO accounts for the activity of endothelium-derived relaxing factor.^{4 5} NO is a potent vasodilator and possesses many antiatherogenic properties. NO decreases platelet aggregation and adhesion,^{6 7} limits vascular smooth muscle proliferation,⁸ inhibits neointima formation,⁹ prevents monocyte chemotaxis,¹⁰ and inhibits leukocyte adhesion to the endothelium.¹¹ NO is generated by the NOS family of proteins,¹² referred to as nNOS (NOS1), iNOS (NOS 2), and eNOS (NOS 3). These enzymes are distinct gene products that catalyze the five-electron oxidation of L-arginine to NO by using flavins, BH₄, and Ca²⁺/calmodulin as critical cofactors and NADPH and molecular oxygen as cosubstrates. In ECs, regulation of NO signaling occurs largely at the level of eNOS activity controlled by cofactors,^{13 14} phosphorylation,^{15 16 17} and targeting of eNOS to specific intracellular membranes by fatty acylation.^{18 19}

We have been interested in the effects of E₂ on EC function and have demonstrated that cytokine-mediated induction of adhesion-molecule gene transcription can be inhibited by E₂ in vitro.²⁰ This is one potential antiatherogenic effect of estrogen. Considerable animal model evidence suggests that NO mediates the vasodilatory effects of E₂. Endothelium-dependent suppression of serotonin- and phenylephrine-induced contraction is greater in thoracic aortic rings isolated from female than from male rats.²¹ Similarly, aortic rings isolated from female rabbits exhibit greater basal NO release than do rings from male or oophorectomized female rabbits.²² Endothelium-dependent vasodilation in oophorectomized nonhuman primates is augmented in animals on estrogen replacement therapy.³ Recently, endothelium-dependent coronary and peripheral vasodilation in response to E₂ has been demonstrated in humans, although the E₂ levels required to achieve these vasomotor responses remain controversial.^{23 24}

Investigations of the effects of E₂ on eNOS activity and NO release in cultured ECs have been conflicting.^{25 26 27} This may be attributable to the wide range of culture conditions employed (age, gender, and species of the cells studied) as well as the assays used for evaluation. We used a phenol red–free gonadal hormone–free HUVEC culture medium to determine whether E₂ has detectable effects on eNOS activation and NO release. In the present study, we demonstrate that in ER-positive female HUVECs, short-term pretreatment with physiological concentrations of E₂ dramatically increases eNOS activity independent of cytosolic Ca²⁺ mobilization, resulting in augmentation of basal NO release. This E₂-stimulated activity appears ER dependent, but transcription independent, and may contribute to the beneficial effect of estrogen in the prevention of coronary artery disease.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials and Methods
E₂, 17-estradiol, GHS, bovine brain calmodulin, DRB, and all other reagents, unless specified, were purchased from Sigma Chemical Co. The ER antagonist ICI 164,384 was a kind gift from A.E. Wakeling, ZENECA Pharmaceuticals, Cheshire, England. BH₄ was purchased from B. Schircks Laboratory. L-Arginine-[guanido-³H] (specific activity, 57.8 mCi/mmol) was purchased from NEN Research Products. Leupeptin was obtained from Boehringer-Mannheim Corp. HEPES was purchased from American Bioanalytical, and fluo 3-AM was from Molecular Probes. A (¹²⁵I) S-GMP-TME radioimmunoassay kit was purchased from Biomedical Technologies Inc.

Cell Culture
HUVECs were isolated from female single-donor umbilical veins as previously described.²⁸ HUVECs were serially passaged on gelatin-coated flasks in phenol red–free DMEM (GIBCO) containing 15% heat-inactivated GHS and supplemented with penicillin (100 µg/mL), streptomycin (100 µg/mL), L-glutamine (2 mmol/L), porcine heparin (100 µg/mL), and EC growth factor (50 µg/mL). Cells were used within five passages and were identified as endothelial by their characteristic cobblestone morphology and presence of factor VIII antigen. Forty-eight hours before the experiments, heparin and EC growth factor were removed from the medium. ECs were treated with E₂ over a range of concentrations and time points, as indicated in "Results" and the figure legends. All samples were harvested in HBSS supplemented with CaCl₂ (1.2 mmol/L), MgSO₄ (0.6 mmol/L), and L-arginine (100 µmol/L) (modified HBSS) in which, unless otherwise specified, cells were incubated for 1 hour before supernatant collection. Ca²⁺-mobilizing agonist treatments were all performed in modified HBSS for 1 hour.

Determination of HUVEC NO_X Release
NO_X, defined as NO₂^-, NO₃^-, and nitrosothiols, was determined by NO-specific chemiluminescence, as previously described.¹⁸ Briefly, HUVECs were grown in the absence or presence of E₂, after which the medium was changed to modified HBSS, as noted above, and equilibrated for 1 hour at 37°C. Agonist treatment (thrombin, 5 U or 50 U; histamine, 5 µmol/L or 20 µmol/L; or ionomycin, 500 nmol/L) was for 1 hour at 37°C. In some experiments, treatment (E₂ or agonists) was performed in the presence of the ER antagonist ICI 164,384 (10 µmol/L) or with L-NMMA (1 mmol/L) present during the 1-hour incubation with modified HBSS. At the end of the incubation period, supernatants were collected for NO_X analysis, and 100 µL was refluxed in heated 0.1 mmol/L vanadium (III) chloride in 2 mmol/L HCl. NO₂^-, NO₃^-, and nitrosothiols are quantitatively reduced to NO_X under these conditions and NO was quantified after reaction with ozone by a chemiluminescence detector (Sievers).

Ca²⁺ Measurements
All Ca²⁺ measurements were performed on an ACAS 570 interactive laser cytometer using a 488-nm argon source (Meridan, Inc). Relative [Ca²⁺]_i was followed using the fluorescent Ca²⁺ probe fluo 3-AM as previously described²⁹ ; this fluorescein-based dye has a peak emission wavelength of 530 nm when excited at 488 nm and gains fluorescence intensity proportionally with increasing [Ca²⁺]_i. HUVECs were plated onto 35-mm dishes and cultured as previously described. The HUVECs were loaded with fluo 3 (final concentration, 2 µmol/L) for 45 to 60 minutes using 0.5% pluronic acid at 22°C, washed twice, and placed in KRH (125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L KH₂PO₄, 1 mmol/L MgSO₄, 2 mmol/L CaCl₂, 25 mmol/L HEPES, and 6 mmol/L glucose) containing 5% GHS. Tissue culture dishes were placed onto the stage of the ACAS 570 and were visualized initially with an integrated inverted microscope under phase contrast to select an appropriate field. Repeated laser excitation of a field (which generally contained four or five HUVECs) was performed with continuous recording of fluorescence data. Scanning before stimulus provided background Ca²⁺ levels, and histamine provided a positive control. Fluorescence intensity curves for individual ECs were generated with the Kinetics software data analysis program available with the ACAS.

NOS Activity in Cell Lysates
NOS activity was assayed as previously described.³⁰ Subconfluent HUVEC monolayers grown in T-75 culture flasks were treated with E₂ vehicle, E₂ (50 ng/mL), or E₂ (50 ng/mL) in the presence of ICI 164,384 (10 µmol/L) for 1 or 24 hours. At the end of the indicated treatment periods, cells were suspended by brief treatment with trypsin-EDTA, washed with PBS, and used to prepare total cell lysate for measurement of eNOS activity.

To prepare total cell lysates, HUVECs were suspended in cold lysis buffer (Tris-HCl [pH 7.5], 50 mmol/L; NP-40, 1% [vol/vol]; EDTA, 0.1 mmol/L; EGTA, 0.1 mmol/L; mercaptoethanol, 0.1% [vol/vol]; NaF, 10 mmol/L; Na₃VO₄, 1 mmol/L; leupeptin, 100 mmol/L; aprotinin, 2 mg/mL; soybean trypsin inhibitor, 10 mg/mL; and phenylmethylsulfonyl fluoride, 1 mmol/L) and rocked at 4°C for 2 hours. To analyze eNOS activity, cell lysates (200 µg) were incubated with NADPH (1 mmol/L), CaCl₂ (2.5 mmol/L), calmodulin (100 nmol/L), L-arginine-HCl (10 µmol/L), [³H]L-arginine (66 Ci/mmol, 0.2 mCi), and BH₄ (30 µmol/L) for 60 minutes at 37°C. Ca²⁺/ calmodulin-independent eNOS activity was measured in replicate samples by omitting CaCl₂ and calmodulin from the incubation. BH₄ dependence was assessed in replicate samples with BH₄ omitted. In all experiments, replicate incubations were performed in the presence of the NOS inhibitor L-NAME (1 mmol/L), and the data were presented as L-NAME–inhibitable conversion of L-arginine to L-citrulline. Incubations were terminated by adding 1 mL of ice-cold stop buffer (20 mmol/L HEPES, 2 mmol/L EDTA, and 2 mmol/L EGTA, pH 5.5). [³H]L-Arginine was separated from [³H]L-citrulline by passing the entire reaction mix over a column containing 1 mL of equilibrated Dowex cation exchange resin. The effluent containing [³H]L-citrulline was collected and quantified by liquid scintillation counting in Ecoscint (National Diagnostics) using a Tricarb liquid scintillation analyzer (model 1500, Packard Instrument Co, Inc). Protein concentration of cell lysates was determined according to the method of Bradford, using bovine serum albumin as a standard.

Determination of HUVEC NO_X Release in the Presence of a Transcriptional Inhibitor
Briefly, HUVECs were grown in the absence of E₂, after which the medium was changed to modified HBSS. The agonist (histamine, 5 µmol/L) or E₂ (10 ng/mL) was added for 1.5 hours at 37°C. In some experiments, treatments (E₂ or agonist) was performed in the presence of the transcriptional inhibitor DRB (20 µg/mL), as previously described.³¹ Inhibition of transcription by DRB was demonstrated in duplicate interleukin-1–treated samples by abrogation of [³H]uridine incorporation (ß-counting) and membrane E-selectin induction (cytofluorimetric analysis). At the end of the incubation period, supernatants were collected for NO_X analysis as described above.

Determination of Intracellular cGMP Concentration
cGMP accumulation in HUVECs was determined using various nitrovasodilators and E₂ with a method previously described.³² Briefly, cells were washed with HBSS to remove serum and incubated with HBSS containing E₂ (10 ng/mL), histamine (5 µmol/L), or sodium nitroprusside (100 µmol/L) for 10 minutes in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (0.3 mmol/L) to prevent cGMP breakdown. Pretreatment of the cells with the L-arginine analogue L-NAME (1 mmol/L) or the ER antagonist ICI 164,384 (10 µmol/L) for 30 minutes was performed in some samples. After the 10-minute incubation, medium was rapidly aspirated, and 200 µL of 0.1N HCl was added to each well to stop enzymatic reactions and extract cGMP. Thirty minutes later, the HCl extract was collected, and the cell remnant was removed from the wells by adding hot 1.0N NaOH and scraping the wells. The HCl extract was analyzed for cGMP by radioimmunoassay, and NaOH-solubilized samples were used for protein determination. Standard and experimental displacement curves were generated as described³² to allow determination of cellular cGMP content.

Data Analysis
Each experiment was performed at least three times, with each data point performed in triplicate. Results are expressed as mean±SE. Significance of results was determined with INSTAT 2.0 data analysis software (Macintosh). Comparisons were performed with Student's t test for unpaired data.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
E₂ Effect on EC NO Release
We have previously demonstrated that HUVECs express ERs and respond to E₂.²⁰ To evaluate whether E₂ pretreatment could modulate HUVEC NO release, cells were propagated in gonadal hormone–free medium and pretreated with E₂ or vehicle for 24 hours followed by a change to modified HBSS. Fig 1 displays the comparative NO_X release, measured by NO-specific chemiluminescence, in E₂ versus traditional Ca²⁺-mobilizing agonist-treated HUVECs. E₂ augments basal NO release in HUVECs, inducing levels comparable to those produced in response to thrombin, ionomycin, or histamine. Pretreatment with 17-estradiol did not augment basal NO_X release (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 1. Comparative effects of E2 and Ca2+-mobilizing agonists on EC NOX release. HUVECs propagated in gonadal hormone–free medium were treated with E2 (50 ng/mL) or vehicle (C) for 24 hours, followed by a median change to modified HBSS for 1 hour. Alternatively, HUVECs were stimulated with thrombin, ionomycin, or histamine at the indicated concentrations in modified HBSS for 1 hour. NO release was measured by chemiluminescence (pmol/3x 106 cells) as described in "Materials and Methods." Data represent mean±SE from eight experiments. *P<.05 vs control.

Dose-response experiments were performed, with concentrations of E₂ ranging from those commonly used in in vitro experiments (1 ng/mL) to concentrations well above the physiological range (200 ng/mL). Fig 2A demonstrates that a 24-hour pretreatment with E₂ (1 ng/mL) achieved >80% of maximally measured NO_X induced with 100 ng/mL. Concentrations as low as 500 pg/mL also augmented basal NO release (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 2. Dose-response relation and kinetics of E2-mediated EC NOX release. A, HUVECs were treated with E2 at the indicated concentrations or E2 vehicle for 24 hours, followed by medium change to modified HBSS for 1 hour and NOX measurement. Cell-free E2 in modified HBSS did not give a chemiluminescent signal at any E2 concentration. B, HUVECs were treated with E2 (50 ng/mL) for the indicated times, followed by medium change to modified HBSS for 1 hour and NOX measurement. Data represent mean±SE from four experiments. *P<.05 vs control (0 ng/mL E2 or 0 hours).

Kinetics of EC NO release to E₂ were investigated, in part to begin approaching the phenomenon from a mechanistic standpoint. HUVECs were treated for the indicated times (Fig 2B) with E₂ (50 ng/mL), washed, and changed to supplemented HBSS for 1 hour, followed by NO_X measurement. Marked augmentation of NO release was demonstrated at the earliest time point (15 minutes) and was maximal after a 1-hour pretreatment. The 15-minute time point actually represents a total of 75 minutes after the onset of E₂ exposure, because of the 60-minute equilibration in HBSS after completion of the stimulus. The 1-hour NO_X levels were identical to those obtained after a 24-hour incubation with E₂. When HUVECs were E₂-treated for 1 hour, washed, and analyzed for NO release 24 hours later, measured NO_X was equal to control without E₂ exposure (data not shown). These results are consistent with prior reports of rapid vascular responses to E₂ and display a requirement for the presence of E₂ in the medium for the observed effect.

To further determine whether transcription is required for the effects of E₂, NO release assays (NO_X measurements) were performed on HUVECs treated with the RNA polymerase II inhibitor DRB. Inhibition of transcription was confirmed in duplicate samples by (1) inhibition of [³H]uridine incorporation and (2) (as a positive control for inhibition of transcription) by abrogation of membrane E-selectin induction in interleukin-1–treated cells (data not shown). Measurable NO_X was identical in DRB-treated or -untreated E₂-stimulated HUVECs (Fig 3A). DRB treatment had no effect on histamine-mediated NO release, as expected. The rapid kinetics of NO release suggest a nongenomic effect of E₂. Because 75 minutes was the shortest time point performed in the NO_X assays (as a result of the required equilibration period), another series of experiments was performed in which L-NAME–inhibitable cGMP generation, a bioassay for NO production, was measured after a 10-minute E₂ treatment of HUVECs. E₂ induced a 15-fold increase in measurable cGMP, similar to that induced by histamine (Fig 3B). As in all the other assays, this E₂ effect was largely inhibitable by L-NAME or ICI 164,384, as expected. These DRB inhibitor and cGMP formation experiments confirm that the control point for ER-mediated NO release is not at the transcriptional level.

View larger version (19K): [in this window] [in a new window]   Figure 3. Role of transcription in E2-mediated EC NO release. A, HUVECs were treated with E2 (10 ng/mL) or histamine (5 µmol/L) in the presence or absence of DRB (20 µg/mL) for 1.5 hours, followed by a medium change to modified HBSS for 1 hour and NOX measurement. Data represent mean±SE. *P<.05 vs control. B, HUVECs were pretreated for 30 minutes with medium control, L-NAME (1 mmol/L), or ICI 164,384 (10 µmol/L), followed by treatment with medium control, E2 (10 ng/mL), or histamine (5 µmol/L) for 10 minutes. HCl (0.1N) was added to each sample to stop the enzymatic reactions and extract cGMP. cGMP was measured by radioimmunoassay, as in "Materials and Methods," and recorded as pmol/mg total protein. Data represent mean±SE. *P<.05 vs L-NAME within each group.

E₂ Effect on EC Cytosolic Ca²⁺
Because E₂ has been shown to facilitate Ca²⁺ entry and mobilization,³³ Ca²⁺ flux experiments were performed on single fluo 3–loaded adherent HUVECs. Fig 4 demonstrates representative Ca²⁺ flux curves in histamine- and E₂-treated cells. Whereas there is the expected rapid rise in [Ca²⁺]_i in response to histamine (Fig 4A), E₂ has no effect on relative [Ca²⁺]_i, either with a single exposure to 50 ng/mL (Fig 4B) or 1000 ng/mL (Fig 4C) E₂ at time 0 or with an exposure to 50 ng/mL E₂ after a 24-hour pretreatment with E₂ at the same concentration (Fig 4D). Despite the lack of Ca²⁺ mobilization induced by E₂, the E₂-treated cells were all responsive to traditional Ca²⁺-mobilizing agents, as demonstrated by their normal responses to histamine after E₂ exposure. Thus, E₂ does not augment NO release in HUVECs by mimicking Ca²⁺-mobilizing agonists.

View larger version (17K): [in this window] [in a new window]   Figure 4. E2 effect on EC [Ca2+]i. HUVECs were grown in gonadal hormone–free medium, followed by a single exposure to histamine (20 µmol/L) (A), E2 (50 ng/mL) (B), E2 (1000 ng/mL) (C), or 24-hour treatment with E2 (50 ng/mL), followed by a repeat exposure to E2 (50 ng/mL) (D). Representative smoothed curves of normalized relative fluorescence in single fluo 3–loaded HUVECs are shown. Histamine-induced Ca2+ fluxes in E2-stimulated cells demonstrate EC responsiveness. Data represent three separate experiments.

E₂ Effect on eNOS Activity
Upon determining that E₂ augments EC NO release, eNOS activity assays were performed to evaluate modulation of enzymatic function and/or eNOS cofactor requirements. L-NAME–inhibitable eNOS activity (conversion of [³H]L-arginine to [³H]L-citrulline) was assayed in whole-cell lysates from control and E₂-treated HUVECs in the presence or absence of exogenous BH₄ or Ca²⁺/calmodulin. Fig 5 demonstrates a comparable requirement for BH₄ in E₂-treated versus -untreated ECs. However, eNOS activity in control cell lysates was dependent on the presence of Ca²⁺/calmodulin. In contrast, these cofactors, generally believed to be essential for eNOS activity, were not required in lysates from E₂-treated cells. This "conversion" from a traditional Ca²⁺/calmodulin-dependent activity to that independent of additional Ca²⁺/calmodulin was seen at early (1-hour) and late (24-hour) time points of E₂ treatment and was inhibited by the ER antagonist ICI 164,384.

View larger version (34K): [in this window] [in a new window]   Figure 5. E2 effect on eNOS activity. Cultured HUVECs were treated with vehicle control (C), E2 (50 ng/mL), or E2 (50 ng/mL) in the presence of ICI 164,384 (ICI, 10 µmol/L) for 1 or 24 hours, after which cell lysates were harvested and incubated with [3H]L-arginine and cofactors as detailed in "Materials and Methods." The terminated reaction mix was passed over a Dowex cation exchange resin, and [3H]L-citrulline was recovered from the effluent, followed by liquid scintillation counting. BH4 or CaCl2/calmodulin (calcium/CAM) was omitted from some lysates to determine the dependence on these cofactors. Data are shown as picomoles L-NAME–inhibitable [3H]L-citrulline per milligram recovered protein. Samples were run in duplicate, and data represent the mean±SE from three experiments. *P<.05 vs all-cofactor sample within group. The (-) calcium/CAM sample was not statistically different from the all-cofactor sample within the E2 (1 hour) or E2 (24 hour) groups.

Effect of Inhibitors on E₂-Mediated EC NO Release
A series of experiments was performed to ascertain ER involvement and eNOS specificity in the noted E₂ responses. Fig 6 demonstrates that the ER antagonist ICI 164,384 significantly inhibits E₂-mediated NO release (70% inhibition in the 24-hour E₂-treated sample), whereas it had no effect on the NO release by histamine. This result confirms that E₂-mediated basal NO augmentation proceeds via hormone receptors. Furthermore, effective inhibition of E₂-induced NO release was observed with the NOS inhibitor L-NMMA (Fig 7) and was equal to that achieved by L-NMMA in the setting of thrombin treatment. This reduction is consistent with the previously reported inhibitor potency of L-NMMA and confirms that E₂-mediated induction proceeds via the L-arginine/NO pathway.

View larger version (24K): [in this window] [in a new window]   Figure 6. ER requirement for E2-mediated EC NOX release. HUVECs were treated with E2 vehicle (control [C]), E2 (50 ng/mL), or histamine, (HIST, 20 µmol/L) in the absence or presence of ER antagonist ICI 164,384 (ICI, 10 µmol/L) for 1 or 24 hours. NOX release was measured after a medium change to modified HBSS for 1 hour. Data represent mean±SE from four experiments. *P<.05 vs ICI 164,384 within each group.

View larger version (13K): [in this window] [in a new window]   Figure 7. Effect of NOS inhibitor on E2-mediated NOX release. HUVECs were treated with E2 vehicle (C) or E2 (50 ng/mL) for 24 hours. After changing the medium to modified HBSS, L-NMMA (1 mmol/L) was added for 20 minutes, followed by the addition of E2 (50 ng/mL) or thrombin (50 U/mL). The samples were harvested at 1 hour, and NOX release was measured. Data represent mean±SE from three experiments. *P<.05 vs L-NMMA within each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The relationship between NO and the cardiovascular protective effects of estrogen, although suggested by several animal studies, is incompletely understood. In the present study, we demonstrate that E₂ pretreatment of HUVECs results in augmented eNOS activity independent of cytosolic Ca²⁺ mobilization, resulting in an increased basal release of L-arginine–derived NO. These data are consistent with E₂ increasing NO-mediated basal coronary arterial diameters in humans^{23 24} and basal NO-dependent relaxation of female rabbit aortic rings.²² Furthermore, a recent report documented a link between ER-mediated responses and basal NO release.³⁴ In that report, basal release of endothelium-derived NO, as determined by endothelium-dependent contraction caused by L-NAME, was markedly reduced in the aorta of homozygous ER knockout mice. In contrast, acetylcholine-induced endothelium-dependent relaxation was similar in the aorta from wild-type and ER knockout mice. Previous reports examining the influence of E₂ on EC NO release in vitro have been both inconclusive and conflicting, largely because of nonstandardized culture conditions, the exact nature of which is critical in evaluation of steroid hormone responses. Recently, we have established in vitro conditions²⁰ by use of single-donor female HUVECs propagated in phenol red–free GHS-containing medium and by which reproducible ER-mediated endothelial responses to E₂ can be observed.

A 48-hour HUVEC treatment with E₂ results in a 2-fold increase in eNOS mRNA levels (Reference 35³⁵ , and T. Caulin-Glaser, W.C. Sessa, J.R. Bender, unpublished data, 1997). Although the eNOS promoter does contain multiple partial estrogen response elements,³⁶ the effects of estrogen demonstrated in the present study are not transcriptionally based, because augmented NO release is maintained in transcription-arrested ECs. Furthermore, the rapid kinetics of E₂-mediated NO release are more consistent with a nongenomic action of the hormone. This is supported by the experiments demonstrating a rapid increase in HUVEC cGMP levels after a 10-minute E₂ treatment. We evaluated the ability of E₂ to raise HUVEC [Ca²⁺]_i as has been shown in other cell types³⁷ and found that it does not. The predominant effect of E₂ on HUVEC NO release appears to be a change in eNOS enzymatic function, mediated via the ER. Surprisingly, cell extracts from E₂-pretreated cells supported the conversion of L-arginine to L-citrulline in the absence of Ca²⁺ or additional calmodulin. This was not due to iNOS activity, as the Ca²⁺-independent enzyme could not be detected at the mRNA or protein level in these HUVECs (T. Caulin-Glaser, W.C. Sessa, J.R. Bender, unpublished data, 1997). We have previously reported that all detectable NOS activity in cytokine-treated HUVECs is Ca²⁺-dependent.¹⁴ Recently, Ca²⁺-independent NOS activity induced in the rat kidney by pregnancy has been demonstrated.³⁸ This activity was not inhibited by conventional arginine analogues, and iNOS protein was undetectable.

With two exceptions, eNOS-derived NO release has been shown to be Ca²⁺/calmodulin-dependent in vitro. The first exception has been the finding that flow-induced³⁹ and/or shear stress–induced^{40 41} NO release may proceed via a Ca²⁺-independent pathway both in situ³⁹ and in vitro.^{40 41} In both intact blood vessels and cultured ECs, the initial phase of NO release induced by flow appears to be Ca²⁺/calmodulin dependent, whereas the sustained shear-induced NO release is independent of Ca²⁺. Although the mechanism(s) of this Ca²⁺ independence was not established, tyrosine kinase inhibition abrogated the shear stress–induced NO release in intact blood vessels.³³ The second exception was that insulin-like growth factor-1 increases HUVEC and rat renal artery EC NO release by a Ca²⁺-independent mechanism. As with shear-induced NO release, this response was also attenuated by tyrosine kinase inhibitors.⁴² Mechanotransduction induced by flow or shear across the EC membrane has been demonstrated to activate normally Ca²⁺-dependent enzymes, such as mitogen- activated protein kinase, in a Ca²⁺-independent manner.⁴³ Although speculated to involve cytoskeletal proteins and a change in eNOS microenvironment, the mechanism by which shear activates eNOS and the role tyrosine kinases play in this activation remain unclear. Studies are currently under way to assess whether induced tyrosine kinase activity may be involved in ER-mediated NO release.

One of the properties of estrogen that is thought to contribute to its cardiovascular protective effects is its antioxidant activity. A recent report demonstrated that ethynylestradiol, through the reduction in superoxide anion generation, augmented endothelium-derived guanylate cyclase–activating activity.⁴⁴ This prevention of NO breakdown by ethynylestradiol may be one critical determinant of the vasodilatory role of estrogen in vivo. However, our results demonstrate an E₂-mediated increase in eNOS activity and quantifiable basal NO release, neither of which were observed in response to ethynylestradiol in this recent report. This difference, taken together with the fact that 17-estradiol (an effective antioxidant) does not augment HUVEC basal NO release, suggests that the antioxidant property of E₂ is not the primary mechanism whereby E₂ exerts the noted effects on NOS activity and NO release.

In summary, the present study demonstrates that E₂ treatment of HUVECs, in vitro, rapidly induces eNOS activity and NO release, in the absence of cytosolic Ca²⁺ mobilization, in an ER-dependent fashion. This raises fascinating questions regarding hormonal regulation of eNOS, including the potential effect of estrogen on eNOS posttranslational modification and localization. It also suggests a potential mechanism whereby estrogen exerts it cardiovascular protective effect.

	Selected Abbreviations and Acronyms

 

BH4 = (6R,S)-5,6,7,8-tetrahydro-L-biopterin DRB = 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole E2 = 17ß-estradiol EC = endothelial cell eNOS, iNOS, nNOS = endothelial, inducible, and neuronal NOS ER = estrogen receptor GHS = gelding horse serum HUVEC = human umbilical vein EC L-NAME = NG-nitro-L-arginine methyl ester L-NMMA = NG-monomethyl-L-arginine NOS = NO synthase NOX = reduced NO2- and NO3-

	Acknowledgments

 
This study was supported by National Institutes of Health grants R01 HL-43331 (Dr Bender), K08 HL-03372-02 (Dr Caulin-Glaser), and R29 HL-51948 (Dr Sessa); the Raymond and Beverly Sackler Foundation Inc; and the Catherine Weldon Donaghue Medical Research Foundation. Dr Bender is a Raymond and Beverly Sackler Foundation Scholar. Dr Sessa is an Established Investigator of the American Heart Association. We express gratitude to Lynn O'Donnell, Louise Benson, and Gwen Davis for assistance with cell culture. We thank all those who provided generous gifts of valuable reagents. We are grateful to the Milford General Hospital Delivery Room nursing staff for obtaining umbilical cords.

Received July 10, 1997; accepted September 17, 1997.

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