Estrogen retards the development of coronary heart disease. The incidence of coronary heart disease is relatively low among premenopausal women and increases sharply with the occurrence of menopause (1,2). Whereas the beneficial effect of estrogen on cardiovascular protection can be attributed in part to its effect on plasma lipoprotein metabolism, studies also have clearly demonstrated that estrogen promotes vasodilation by directly acting on endothelium and vascular smooth muscle (3,4).
Studies in postmenopausal women showed that physiologic levels of estrogen potentiate vasodilation to acetylcholine in large conductance as well as coronary microvascular resistance arteries (5-7). Animal studies demonstrated that long-term estrogen replacement decreased contractility of guinea pig coronary arteries to the thromboxane mimetic U46619 (8). The authors suggested that an increase in nitric oxide (NO) release and cyclic guanosine monophosphate (cGMP) levels was involved in the estrogen-mediated effect. Studies in cultured noncoronary endothelial cells indicated that estrogen increased NO release through genomic as well as nongenomic manners (9-15). Depending on the cells studied, conflicting data are available on the cellular mechanisms mediating estrogen action on endothelial nitric oxide synthase (eNOS) activity and NO release in cultured endothelial cells. For instance, studies in endothelium from human umbilical vein, aorta, bovine aorta, and sheep fetal pulmonary artery demonstrated that 17β-estradiol (E2β) increases NO release and eNOS protein levels (9-11). However, studies by Arnal et al. (16) in bovine aortic endothelium showed that ethinylestradiol, a stable analogue of E2β, had no effect on either the activity or expression of eNOS despite its ability to increase NO release. In addition, a recent study demonstrated that the E2β-mediated increase in NO release in human umbilical vein endothelium was independent of cytosolic Ca2+ mobilization and transcription of eNOS (12).
To our knowledge, no study has yet determined the direct effect of estrogen on NO release in coronary artery endothelial cells despite its well-known protective effect on the coronary circulation. Given the heterogeneous nature of the endothelium from different vascular beds, our study was thus designed to test the hypothesis that estrogen increased NO release in human coronary artery endothelial cells (HCAECs). To understand cellular mechanisms, we also determined the effect of estrogen on cytosolic Ca2+ mobilization and on eNOS protein expression in HCAECs.
Third-passage HCAECs were obtained from Clonetics Co. (Walkersville, MD, U.S.A.). The cells were seeded at a density of 5,000/cm2 in endothelial cell basal medium containing human endothelial growth factor (hEGF; 10 ng/ml), gentamicin (50 μg/ml)/amphotericin-B (50 ng/ml), hydrocortisone (1 μg/ml), bovine brain extract (12 μg/ml), and fetal bovine serum (20%), and cultured at 37°C in a humidified incubator with 5% CO2 in air. The cells at the fifth and sixth passages with ∼80% confluence were placed in phenol red-free serum-free medium for 24 h to remove the effect of estrogen in the medium. The cells were then cultured in phenol red-free medium containing 16% charcoal-stripped serum in the absence or presence of E2β and 17α-estradiol (E2α) for 48 h. To determine the role of estrogen receptors in E2β-mediated effects, some experiments were performed in the presence of estrogen receptor antagonist ICI 182,780. Estrogens and ICI 182,780 were dissolved in dimethylsulfoxide (DMSO; 0.01% final concentration in the treatment), and the control media contained the same concentration of DMSO.
Measurement of NOx
NO was measured by chemiluminescence method as described previously (17). Because of the instability of NO in physiologic solution, most of NO is rapidly converted to nitrite and further to nitrate. Nitrite and nitrate are relatively stable in the solution and are readily reduced back to NO in vanadium (III)/HCl solution. The samples (100 μl) taken from the medium were injected into the gas purge vessel containing 5 ml vanadium (III)/HCl and allowed to react for 1 min and reduce nitrate/nitrite in the sample back to NO. To achieve high reducing efficiency, the reduction was performed at 90°C. NO in the sample was then "stripped" into the head space of the gas purge vessel by bubbling it with helium (12 ml/min) for 60 s. NO in the head space was drawn into a NO Analyzer (model 270B; Sievers Instruments, Inc., Boulder, CO, U.S.A.) and mixed with ozone (O3) in front of a cooled Hamamatsu, redsensitive photomultiplier tube. Signals from the detector were analyzed by an on-line computer as area under the peak. The measurement reflected the combined concentrations of nitrate, nitrite, and nitric oxide (NOx) of each sample, which was calculated from a standard curve of 10-1,000 picomoles nitrate run in each assay.
Measurement of intracellular free Ca2+ concentration ([Ca2+]i)
[Ca2+]i was measured in single cells as previously described (18). In brief, after the treatment, the cells were loaded with fura-2 by incubation with 5 μM fura-2/AM for 45 min at 37°C in the loading buffer containing NaCl, 125 mM; KCl, 5 mM; KH2PO4, 1 mM; MgSO4, 1 mM; CaCl2, 2 mM; HEPES, 25 mM (pH 7.4); D-glucose, 6 mM; neostigmine, 10 nM; and Cremophor EL, 0.02%. The cells were then washed 3 times and incubated in Krebs solution for 15 min at 37°C to allow complete hydrolysis of the ester groups by endogenous esterases. Fura-2 fluorescence was monitored photometrically at an emission wavelength of 510 nm in a single cell mounted on a Nikon Diaphot inverted microscope illuminated alternatively with 340- and 380-nm light using an InCyt Im2 Intracellular Imaging System (Intracellular Imaging Inc., Cincinnati, OH, U.S.A.). Photon counting was performed by a photomultiplier tube positioned so that the field of interest may be restricted by thresholding shutters. Data acquisition was accomplished with the software that controls a light chopper to alternate excitation wavelengths during rationing operations. [Ca2+]i was calculated in real time from a standard curve established for the same settings using buffers of known Ca2+ concentrations.
Western analysis of NO synthase
After the treatment, the endothelial cells were solubilized by sonication in lysis buffer (NaCl, 150 mM; Tris HCl, 50 mM; EDTA, 10 mM; Tween 20, 0.1%; β-mercaptoethanol, 0.1%; phenylmethylsulfonyl fluoride, 0.1 mM; leupeptin, 5 μg/ml; and aprotinin, 5 μg/ml; at pH 7.4). After centrifugation, protein was quantified in the supernatant by the method of Bradford (19). Samples with equal protein (10 μg) were loaded on a 7.0% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) and were separated by electrophoresis at 100 V for 2 h. Proteins were then transferred onto immobilon P membrane at 30 V for 30 min at room temperature using a semidry blotter (Bio-Rad). The immobilon P membrane was probed by mouse monoclonal antiserum for endothelial isoform of NO synthase (1:750) obtained from Transduction Laboratories (Lexington, KY). The secondary antiserum was horseradish peroxidase-conjugated goat anti-mouse (1:1,000) obtained from Amersham (Arlington Heights, IL, U.S.A.). Proteins were visualized with enhanced chemiluminescence (ECL) reagents (Amersham), and the blots were exposed to hyperfilm. Results were quantified by scanning densitometer (model 670; Bio-Rad) and expressed as percentage of the control values.
Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad software, San Diego, CA, U.S.A.). Half-maximal effective concentration (EC50) values for an agonist in each experiment were taken as the molar concentration at which the contraction-response curve intersected 50% of the maximal response, and were expressed as pD2 (−log EC50) values. Data are presented as the mean ± SEM. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Newman-Keuls tests. Values were considered statistically significant at p < 0.05.
Effect of E2β on NO release
The effect of E2β on basal NOx release in HCAECs is shown in Fig. 1. The cells were treated with control medium or medium with 36 nM E2β for 48 h. NO (measured as NOx) in the medium was assayed by the chemiluminescence method. Over a 48-h period, basal NOx concentration in the medium of control cells was 546.0 ± 49.5 nM. E2β treatment significantly increased basal NOx release from HCAECs and produced a 2.3-fold increase in NOx concentration in the medium. In contrast, the same concentration of E2α, the less active stereoisomer of estrogen, did not augment basal NOx release in HCAECs. To ascertain the involvement of estrogen receptors, the effect of E2β on NOx release was determined in the absence or presence of 3 nM ICI 182,780, the pure estrogen-receptor antagonist. Whereas ICI 182,780 itself did not have effect on NOx release (data not shown), it blocked the E2β-induced increase in NOx release form HCAECs (Fig. 1).
Figure 2 shows the effect of E2β on calcium ionophore A23187-induced NOx release in HCAECs. The cells were pretreated with either control medium or medium with E2β or E2α (36 nM) for 48 h, and then incubated with increasing concentrations of calcium ionophore A23187 for 1 h. As shown in Fig. 2, calcium ionophore A23187 produced concentration-dependent increases in NOx release in both control and E2β-treated cells. The A23187-induced NOx release was significantly higher in the E2β-treated, but not E2α-treated, cells than that in the control cells. The effect of E2β was blocked in the presence of ICI 182,780. Similar to the findings with calcium ionophore A23187, adenosine triphosphate (ATP)-induced NOx release from HCAECs was significantly elevated at all concentrations in the E2β-treated cells compared with the control cells, which was blocked by estrogen-receptor antagonist ICI 182,780 (Fig. 3).
Effect of E2β on Ca2+ mobilization
Intracellular free Ca2+ concentration ([Ca2+]i) in HCAECs was examined in single cells loaded with fura 2. E2β (36 nM) treatment for 48 h did not change basal [Ca2+]i in HCAECs (73.2 ± 15.9 vs. 76.3 ± 13.9 nM). ATP, by activating P2y purinergic receptors, produced concentration-dependent increases in [Ca2+]i in both control cells (pD2, 5.1 ± 0.2) and E2β-treated cells (pD2, 4.8 ± 0.3; p > 0.05; Fig. 4).
Effect of E2β on eNOS protein expression
Figure 5 shows the effect of E2β on endothelial NO synthase (eNOS) protein expression in HCAECs. The representative Western immunoblot showed that the monoclonal antibody for eNOS detected a single band at the expected size of 145 kDa (Fig. 5, top). There was an enhancement of eNOS protein expression in the E2β-treated cells. Quantitative densitometry for four independent experiments revealed that eNOS protein expression in the E2β-treated cells was increased to 189.3% of the value in the control cells (Fig. 5, bottom).
This study demonstrated for the first time that prolonged E2β treatment increased both basal and agonist-stimulated NOx release in HCAECs. Similar findings were obtained in fetal ovine pulmonary artery endothelium in which E2β treatment for 48 h significantly increased NO synthase activity (9). However, no direct measurement of NO release was done in the earlier study (9). Our results directly support the interpretation that increased NO release in coronary artery endothelium plays a key role in estrogen-induced decrease in basal coronary artery tone and increase in coronary artery blood flow in humans (5-7). The finding that E2α failed to increase NOx release in our study suggests that effect of E2β is specific and mediated by activation of estrogen receptors in HCAECs. The involvement of the classic type-1 estrogen receptors is evident by the finding that ICI 182,780 inhibited E2β-mediated NOx release in HCAECs. It has been demonstrated that ICI 182,780 is a highly specific estrogen-receptor antagonist and inhibits both α and β isoforms of classic estrogen receptors (9,20,21).
Not only was basal NOx release elevated, but agonist-stimulated NOx release also was increased by E2β in HCAECs. In our study, we chose two different agonists to study the effect of E2β on agonist-mediated NO release. The actions of ATP on vascular endothelial cells, leading to release of NO and vasodilatation, have been described in many vessels including coronary artery (22,23). ATP, acting on P2y purinergic receptors, stimulates inositol 1,4,5-triphosphate production and intracellular Ca2+ mobilization, which in turn activates endothelial NO synthase in the endothelium. We have shown that ATP produces concentration-dependent increases in NOx release in HCAECs. Although the increased ATP-induced NOx release by E2β in HCAECs may be due, in part, to increased interactions between ATP and P2y receptors, the finding that calcium ionophore A23187-stimulated NOx release also was significantly enhanced in E2β-treated cells suggests that the postreceptor mechanisms are involved in E2β-mediated increase in agonist-induced NOx release. This is confirmed by the finding that E2β had no effect on ATP-induced increases in [Ca2+]i in HCAECs. Our recent study demonstrated that ATP-stimulated NOx release in uterine artery endothelial cells was significantly higher in pregnant than in nonpregnant animals, which was mediated by an increase in endothelial eNOS protein expression (17).
Whereas this study demonstrated that prolonged estrogen exposure had direct stereospecific effects on HCAECs, resulting in an increase in NOx release, nongenomic actions of estrogen on the endothelium have also been demonstrated (12-15,24). A recent study demonstrated that E2β produced a rapid increase in NO release in human umbilical vein endothelial cells (12). In the coronary vasculature of postmenopausal women, administration of E2β attenuated the acetylcholine-induced vasoconstriction and increased basal blood flow (6,25). Whereas these results were interpreted as an nongenomic action of estrogen on coronary artery endothelium, studies in the isolated perfused rat heart demonstrated that the acute vasodilation actions of estrogen on the coronary vasculature were NO- and endothelium-independent, suggesting a direct effect on underlying smooth-muscle cell membrane (4,26-28). There is considerable evidence that estrogen has an acute effect on vascular smooth muscle and induced relaxation (29-31). Recent studies in guinea pig demonstrated that long-term estrogen replacement decreased contractility of coronary arteries to the thromboxane mimetic U46619 through NO-dependent mechanisms (8). Our finding of increased NOx release by prolonged E2β treatment directly supports the interpretation that genomic actions of estrogen on coronary artery endothelium play an important role in estrogen-mediated hemodynamic effects in the coronary circulation.
NO is synthesized from L-arginine through Ca2+/calmodulin-sensitive NO synthase (eNOS) expressed constitutively in the endothelium. In many studies, the short-term nongenomic stimulation of NO release by estrogen was mediated by an increase in intracellular Ca2+ in the endothelium (13-15,24). However, studies in human umbilical vein endothelium demonstrated that E2β failed to increase [Ca2+]i despite its brief (15 min) effect on NO release (12). To our knowledge, no study has been done to examine the effect of prolonged E2β treatment on Ca2+ mobilization in the endothelium. The finding that 48 h E2β treatment did not change basal [Ca2+]i in HCAECs suggests that E2β-induced increase in basal NOx release is not Ca2+ dependent. Despite the lack of effect of E2β on basal [Ca2+]i in HCAECs, the E2β-treated cells were responsive to traditional Ca2+-mobilizing agent ATP. We previously demonstrated that ATP, by activating P2y receptors, stimulated a dose-dependent increase in [Ca2+]i in guinea pig cardiac endothelial cells (18). In this study, ATP increased [Ca2+]i in both control and E2β-treated HCAECs in a dose-dependent manner, suggesting that the cells retain their normal Ca2+ responses to extracellular stimuli. It is likely that ATP-induced increase in NOx release in HCAECs is mediated by an increase in intracellular Ca2+. The finding that pretreatment of E2β had no effect on ATP-stimulated Ca2+ responses in this study further confirms that the effect of prolonged E2β treatment on NOx release in HCAECs is Ca2+ independent.
The effect of E2β on NOx release in HCAECs is likely to be mediated by the upregulation of eNOS. In this study, 48-h E2β treatment resulted in an increase of eNOS in HCAECs to 190% of the control values. The same finding was obtained in fetal ovine pulmonary artery endothelium (9). It has been well documented that expression of eNOS is regulated by a variety of stimuli. We and others have demonstrated that pregnancy significantly increases eNOS in freshly isolated ovine uterine artery endothelial cells, which is associated with increased NO release (17,32). Estrogen-receptor binding elements were identified in the gene encoding eNOS, suggesting a receptor-mediated action of estrogen on eNOS expression (33,34). It has been demonstrated that E2β treatment (8-48 h) upregulates mRNA levels of eNOS in cultured human umbilical vein endothelium (11). Recent elegant studies by MacRitchie et al. (9) have established that treatment (48 h) of cultured fetal ovine pulmonary artery endothelial cells with E2β increases eNOS activity and upregulates eNOS protein and mRNA expression that is secondary to enhanced eNOS gene transcription.
In summary, we have demonstrated for the first time that prolonged estrogen treatment increases NOx release in HCAECs, which is likely to be Ca2+ independent and be mediated by the upregulation of eNOS protein expression. Whereas the short-term effect of estrogen on NO release in HCAECs has not been examined in this study, it remains a promising area for the future investigation. We speculate that both genomic and nongenomic actions of estrogen complement each other and play an important role in modulating coronary artery endothelial function. This is evident in recent studies of fetal ovine pulmonary artery endothelium in which both genomic and nongenomic actions of estrogen have been identified (9,24).
Acknowledgment: This work was supported in part by California State University San Bernardino Faculty Development Grant. We thank DaLiao Xiao for his technical assistance.
1. Barret-Connor E, Bush TL. Estrogen
and coronary heart disease in women. JAMA
2. Wenger NK, Speroff L, Packard B. Cardiovascular health and disease in women. N Engl J Med
3. Farhat MY, Lavigne MC, Ramwell PW. The vascular protective effects of estrogen
. FASEB J
4. Ruehlmann DO, Mann GE. Actions of oestrogen on vascular endothelial and smooth-muscle cells. Biochem Soc Trans
5. Collins P, Rosano GM, Sarrel PM, et al. 17 beta-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation
6. Gilligan DM, Quyyumi AA, Cannon RO. Effects of physiological levels of estrogen
on coronary vasomotor function in postmenopausal women. Circulation
7. Williams JK, Adams MR, Herrington DM, Clarkson TB. Short-term administration of estrogen
and vascular responses of atherosclerotic coronary arteries. J Am Coll Cardiol
8. Thompson LP, Weiner CP. Long-term estradiol replacement decreases contractility of guinea pig coronary arteries to the thromboxane mimetic U46619. Circulation
9. MacRitchie AN, Jun SS, Chen Z, et al. Estrogen
upregulates endothelial nitric oxide
synthase gene expression in fetal pulmonary artery endothelium. Circ Res
10. Hayashi T, Yamada K, Esaki T, et al. Estrogen
increases endothelial nitric oxide
by a receptor-mediated system. Biochem Biophys Res Commun
11. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T. Up-regulation of nitric oxide
synthase by estradiol in human
aortic endothelial cells. FEBS Lett
12. Caulin-Glaser T, Garcia-Cardena G, Sarrel P, Sessa WC, Bender JR. 17 Beta-estradiol regulation of human
endothelial cell basal nitric oxide
release, independent of cytosolic Ca2+
mobilization. Circ Res
13. Goetz RM, Thatte HS, Prabhakar P, Cho MR, Michel T, Golan DE. Estradiol induces the calcium-dependent translocation of endothelial nitric oxide
synthase. Proc Natl Acad Sci U S A
14. Kim HP, Lee JY, Jeong JK, Bae SW, Lee HK, Jo I. Nongenomic stimulation of nitric oxide
release by estrogen
is mediated by estrogen
receptor alpha localized in caveolae. Biochem Biophys Res Commun
15. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW. Estrogen
receptor alpha mediates the nongenomic activation of endothelial nitric oxide
synthase by estrogen
. J Clin Invest
16. Arnal JF, Clamens S, Pechet C, et al. Ethinylestradiol does not enhance the expression of nitric oxide
synthase in bovine endothelial cells but increases the release of bioactive nitric oxide
by inhibiting superoxide anion production. Proc Natl Acad Sci U S A
17. Xiao D, Liu Y, Pearce WJ, Zhang L. Endothelial nitric oxide
release in isolated perfused ovine uterine arteries: effect of pregnancy. Eur J Pharmacol
18. Yang S, Buxton IL, Probert CB, Talbot JN, Bradley ME. Evidence for a discrete UTP receptor in cardiac endothelial cells. Br J Pharmacol
19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem
20. Mosselman S, Polman J, Dijkema R. ER beta: identification and characterization of a novel human estrogen
receptor. FEBS Lett
21. Wakeling AE, Dukes M, Bowler J. A potent specific pure antiestrogen with clinical potential. Cancer Res
22. Barnard EA, Burnstock G, Webb TE. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci
23. Burnstock G. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology
24. Lantin-Hermoso RL, Rosenfeld CR, Yuhanna IS, German Z, Chen Z, Shaul PW. Estrogen
acutely stimulates nitric oxide
synthase activity in fetal pulmonary artery endothelium. Am J Physiol
25. Reis SE, Gloth ST, Blumenthal RS, et al. Ethinyl estradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women. Circulation
26. Belfort MA, Saade GR, Suresh M, Vedernikov YP. Effects of estradiol-17 beta and progesterone on isolated human
omental artery from premenopausal nonpregnant women and from normotensive and preeclamptic pregnant women. Am J Obstet Gynecol
27. Jiang C, Sarrel PM, Poole-Wilson PA, Collins P. Acute effect of 17 beta-estradiol on rabbit coronary artery
contractile responses to endothelin-1. Am J Physiol
28. Jiang CW, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Endothelium-independent relaxation of rabbit coronary artery
by 17 beta-oestradiol in vitro. Br J Pharmacol
29. Han SZ, Karaki H, Ouchi Y, Akishita M, Orimo H. 17 beta-Estradiol inhibits Ca2+
influx and Ca2+
release induced by thromboxane A2
in porcine coronary artery
30. Zhang F, Ram JL, Standley PR, Sowers JR. 17 beta-Estradiol attenuates voltage-dependent Ca2+
currents in A7r5 vascular smooth muscle cell line. Am J Physiol
31. Wehling M, Neylon CB, Fullerton M, Bobik A, Funder JW. Nongenomic effects of aldosterone on intracellular Ca2+
in vascular smooth muscle cells. Circ Res
32. Magness RR, Shaw CE, Phernetton TM, Zheng J, Bird IM. Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression. Am J Physiol
33. Venema RC, Nishida K, Alexander RW, Harrison DG, Murphy TJ. Organization of the bovine gene encoding the endothelial nitric oxide
synthase. Biochim Biophys Acta
34. Miyahara K, Kawamoto T, Sase K, et al. Cloning and structural characterization of the human
endothelial nitric-oxide-synthase gene. Eur J Biochem