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Am J Physiol Heart Circ Physiol 285: H2150-H2157, 2003. First published July 24, 2003; doi:10.1152/ajpheart.00966.2002
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Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-{alpha}-deficient mice

Judy M. Muller-Delp,1 Dennis B. Lubahn,2,3 Kathryn E. Nichol,1 Brian J. Philips,2 Elmer M. Price,4,6 Edward M. Curran,2 and M. Harold Laughlin4,5,6

1Department of Health and Kinesiology, Texas A&M University, College Station, Texas 77843; 2Departments of Biochemistry, 3Child Health, 4Biomedical Sciences, and 5Medical Physiology, 6The Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211

Submitted 7 November 2002 ; accepted in final form 21 July 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-{alpha} (ER-{alpha}) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-{alpha} knockout (ER{alpha}KO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ER{alpha}KO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ER{alpha}KO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-{alpha} or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ER{alpha}KO mice. eNOS protein was reduced in ER{alpha}KO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ER{alpha}KO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-{alpha}. Ovariectomy reduced SOD-1 protein in ER{alpha}KO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-{alpha}. NO-dependent responses are preserved in ER{alpha}KO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.

endothelial nitric oxide synthase; superoxide dismutase; acetylcholine; ovariectomy; sodium nitroprusside

IN WOMEN, the risk of coronary heart disease increases significantly after menopause (23, 25). Both the morbidity and the mortality from coronary disease are reduced for young adult females compared with males or postmenopausal females (9, 23, 31). Estrogen is known to play a role in these differences (9, 10). Estrogen therapy reduces the risk of cardiovascular disease in healthy postmenopausal women (9, 10, 23, 24). Estrogen enhances endothelial function of coronary arteries and other large arteries, and this may contribute to the cardioprotective effects of estrogen (3, 30, 31, 33, 36, 38).

The precise mechanisms that mediate of the beneficial effects of estrogen on arterial endothelial function are incompletely understood. Long-term effects of estrogen occur through activation of estrogen receptors and subsequent modulation of gene expression (3, 11, 15, 19, 24). Estrogen has been shown to affect endothelium-dependent function through its effects on expression of endothelial nitric oxide synthase (11, 17, 22, 35). In some models, a specific role for the estrogen receptor in modulation of endothelial nitric oxide (NO) synthase (eNOS) expression and activity has been identified. Aortas from male mice in which estrogen receptor-{alpha} (ER-{alpha}) has been disrupted showed less basal release of NO than aortas from wild-type (WT) mice (28). Similarly, hearts from male knockout mice lacking ER-{alpha} (ER{alpha}KO) demonstrated a depressed flow response and reduced nitrite production during reperfusion after ischemia compared with WT mice (39). However, the role of ER-{alpha} in mediating the availability of NO and NO-dependent vasodilation in the coronary circulation has not yet been investigated.

The purpose of this study was to test the hypothesis that disruption of ER-{alpha} would lead to decreased NO-dependent dilation in coronary arteries. To investigate the role of ER-{alpha} in mediating the effects of estrogen on NO-dependent function, vasoreactivity to acetylcholine and sodium nitroprusside and protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1) were evaluated in coronary arteries from WT mice and ER{alpha}KO mice. To assess the role of alternate estrogen receptors or signaling pathways in mediating the effects of estrogen on coronary artery endothelial function, we also evaluated vascular eNOS and SOD-1 protein content and reactivity of coronary arteries from WT and ER{alpha}KO mice after ovariectomy and ovariectomy with estrogen supplementation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. All procedures performed in this study were approved by the Texas A&M University Laboratory Animal Care Committee and the University of Missouri Animal Care and Use Committee. All methods conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996).

ER{alpha}KO mice were obtained by mating mixed C57BL6/129SV mice that were heterozygous for ER-{alpha} gene disruption, as described previously (21). After genotyping, only homozygous ER{alpha}KO females and homozygous WT C57BL6 females were used in experiments. A total of 43 ER{alpha}KO and 36 WT females were used for the study of coronary artery vasomotor reactivity experiments, and a total of 19 ER{alpha}KO and 18 WT females were used for immunoblot experiments. The average age of ER{alpha}KO mice was 16 ± 1 wk. In WT mice, average age was 15 ± 1 wk. Within the WT group, 16 mice were ovariectomized. Sixteen ER{alpha}KO mice were ovariectomized. Experiments were performed no sooner than 10 days after ovariectomy. Estrogen treatment was initiated after 10 days of rest after ovariectomy. Seventeen of the ovariectomized ER{alpha}KO and eight of the ovariectomized WT mice received subcutaneous implants of a 17{beta}-estradiol (E2) pellet (0.25 mg/21 day release) by incision with a 12-gauge trocar needle or subjected to a sham incision with a trocar needle. E2 treatment continued for 14 days before the mice died and the coronary arteries were harvested.

Isolation of coronary arteries. The mice were anesthetized with pentobarbital sodium (50 mg/kg ip). The chest was opened with a midline thoracotomy, and heparin (1 U/g) was administered by intracoronary injection. The heart was then excised and placed in cold (4°C) physiological saline solution (PSS) containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS buffer, and 1 g/100 ml bovine serum albumin, pH 7.4. With the aid of a dissecting microscope (Olympus SVH10), coronary arteries were dissected free of the surrounding myocardium. The arteries (0.4–1.0 mm in length, 90–220 µm inner diameter) were transferred to a Lucite chamber containing PSS equilibrated with room air. Each end of the artery was cannulated with a micropipette and secured with nylon suture. After cannulation, the vessel chamber was transferred to the stage of an inverted microscope (Olympus IX70) equipped with a video camera (Panasonic BP310), video caliper (Microcirculation Research Institute, Texas A&M University), and data-acquisition system (MacLab/Macintosh) for online recording of intraluminal diameter. Arterioles were initially pressurized to 60 cmH2O with two independent hydrostatic pressure reservoirs. Leaks were detected by pressurizing the vessel, the valves were then closed to the reservoirs, and a constant intraluminal pressure was verified. Arteries that exhibited leaks were discarded. Once pressurized, arteries were warmed to 37 ± 1°C and allowed to develop spontaneous tone. Spontaneous tone development did not differ between groups, and average tone development was 8.8 ± 2.1% and 8.8 ± 7.2% in arteries from WT and ER{alpha}KO mice, respectively. To ensure a similar tone in all vessels before the dilator responses were evaluated, the arteries were then preconstricted by the addition of incremental amounts of endothelin (1/2 log increments beginning with a concentration of 1 x 10–12 M) until the vessel diameter decreased by at least 25%.

Evaluation of vasodilatory responses. Once the arteries displayed a stable level of preconstriction (at least 10 min without significant diameter changes), concentration-response relationships to cumulative addition of acetylcholine (1 x 10–9–3 x 10–5 M) and sodium nitroprusside (1 x 10–10–1 x 10–4 M) were determined. The vessels were allowed to equilibrate at least 30 min and redevelop tone between successive determinations of vasodilatory responses. After completion of the final concentration-response relationship, the vessel was washed twice in calcium-free PSS to obtain a maximal diameter. This solution was similar to PSS-albumin solution except that it contained 2 mM EDTA and CaCl2 was replaced with 2.0 mM NaCl.

Evaluation of eNOS and SOD-1 protein. Coronary arteries were isolated from the myocardium as described above and immediately frozen at –70°C in microcentrifuge tubes. Because there was not sufficient protein in a single mouse coronary artery to allow measurement of protein content and have sufficient sample to run on an SDS gel, it was necessary to pool samples of coronary arteries from three mice into one sample. eNOS and SOD-1 protein content of arteries was determined by loading equal amounts of total artery protein (5 µg protein) from equal numbers of different groups on the same gel allowing comparisons between groups on the same gel (18). Frozen arteries were solubilized in a 20-µl Laemmli buffer composed of 62.5 mM Tris, pH 6.8, 6 M urea, 160 mM DTT, 2% SDS, 0.001% bromophenol blue, which was boiled for 2 min, and then vortexed vigorously. Total protein was measured with NanoOrange Protein Quantitation kits (Molecular Probes), which allows measurement of total protein in small samples. Artery lysates were subjected to SDS-PAGE under reducing conditions, and proteins were transferred to polyvinylidene difluoride membrane (Hybond-ECL, Amersham). The membrane was blocked for 1 h at room temperature with 5% nonfat milk in 20 mM/l Tris · HCl, 137 mM/l NaCl, and 0.1% Tween 20. Blots were incubated overnight (25°C) with a primary antibody against eNOS (1:1,600; Transduction Laboratories), followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse). Subsequently the blots were again incubated overnight with primary antibody against SOD-1 (1:2,500, Stressgen), followed by a 1-h incubation with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-rabbit). Proteins were detected by enhanced chemiluminescence (ECL, Amersham) and evaluated by densitometry (NIH Image software).

Solutions and drugs. Stock solutions of drugs were prepared in distilled water and frozen. Fresh dilutions for use in experimental protocols were prepared daily. Albumin was purchased from USB (Cleveland, OH). Endothelin was purchased from Phoenix Pharmaceuticals (Belmont, CA). E2 pellets were obtained from Innovative Research (Sarasota, FL). All other drugs were purchased from Sigma (St. Louis, MO).

Data analysis. Tone development was expressed as the percent decrease from maximal diameter according to the following formula

where Dm is the maximal diameter recorded at 60 cm H2O and Ds is the steady-state diameter recorded after equilibration of the vessel. Vasodilatory responses were recorded as actual diameters and subsequently expressed as the percentage of maximal relaxation according to the following formula

where Ds is recorded after each addition of the drug and Db is the initial baseline diameter recorded immediately before the first addition of the vasodilatory agent. For concentration-response data, a two-way repeated-measures ANOVA was used to detect differences between and within factors. Post hoc analyses were performed with Bonferroni's test for pairwise comparisons where appropriate. One-way ANOVA was used to determine differences in protein levels between groups, followed by Bonferroni's test for pairwise comparisons. All data are presented as means ± SE. In all statistical analyses, n indicates the number of animals in each group. Significance was defined as P <= 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Vasodilation to acetylcholine. Vasodilation to acetylcholine was maintained in coronary arteries from ER{alpha}KO mice. Contrary to our hypothesis, neither sensitivity nor maximal dilation to ACh was reduced in arteries from ER{alpha}KO mice (Fig. 1). Because E2 levels in ER{alpha}KO female mice have been reported to be as high as 10 times that of WT controls (4), we also evaluated acetylcholine responses in coronary arteries from ER{alpha}KO mice and WT controls after ovariectomy to remove effects of circulating E2. After ovariectomy, vasodilatory responses to acetylcholine tended to be greater in WT mice compared with responses in ER{alpha}KO mice (P = 0.19, WT vs. ER{alpha}KO ovariectomy). Ovariectomy did not alter the acetylcholine responses of coronary arteries from WT mice (Fig. 2A), but it significantly reduced acetylcholine responses in coronary arteries from ER{alpha}KO mice (Fig. 2C). Pharmacological estrogen replacement by implant (circulating E2 levels of ~800 pg/ml) (5) restored responsiveness to acetylcholine in arteries from ovariectomized ER{alpha}KO mice (Fig. 2D) but had no effect on acetylcholine-induced vasodilation in ovariectomized WT mice (Fig. 2B).



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  		Fig. 1. Endothelium-dependent vasodilation to increasing concentrations [1 x 10–9 (1e-9) to 3 x 10–5 (3e–5)] acetylcholine in coronary arteries from wild-type (WT) and estrogen receptor-{alpha} (ER-{alpha}) knockout (ER{alpha}KO) mice. Responsiveness to acetylcholine was not altered by disruption of ER-{alpha} (P = 0.205, KO vs. WT).

 


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  		Fig. 2. Effect of ovariectomy (OVEX) and subsequent estrogen treatment on acetylcholine-induced vasodilation. Ovariectomy did not alter the responses of coronary arteries to acetylcholine in WT mice (A) but significantly reduced responses to acetylcholine in ER{alpha}KO (KO) mice (C). Ovariectomy and subsequent estrogen treatment were without effect on acetylcholine-induced dilation in WT mice (B). 17{beta}-Estradiol (E2) restored dilation in ovariectomized ER{alpha}KO mice to the level of intact ER{alpha}KO mice (D). *P < 0.05, KO vs. KO OVEX.

 

Vasodilation to sodium nitroprusside. Vasodilatory responses to sodium nitroprusside were not altered in coronary arteries from ER{alpha}KO mice compared with WT controls (Fig. 3A). The removal of circulating E2 by ovariectomy did not affect vasodilation to sodium nitroprusside in either ER{alpha}KO mice or WT controls (Fig. 3, B and C).



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  		Fig. 3. Endothelium-independent vasodilation to sodium nitroprusside in coronary arteries was not different in WT and ER{alpha}KO mice (A). Ovariectomy did not alter vasodilation to sodium nitroprusside in WT (B) or ER{alpha}KO (C) mice.

 

Effect of NOS blockade. Blockade of NOS with NG-nitro-L-arginine methyl esther (L-NAME) abolished dilation to acetylcholine in arteries from both WT (Fig. 4A) and ER{alpha}KO mice (Fig. 4B), indicating that this response is mediated by NO even in the absence of ER-{alpha}. eNOS expression. Disruption of ER-{alpha} resulted in a slight but nonsignificant reduction in protein levels for eNOS (P = 0.1501, WT vs. ER{alpha}KO; Fig. 5A). Ovariectomy was performed in ER{alpha}KO mice, and eNOS protein was again evaluated to determine whether E2 exerted an effect on eNOS protein expression through a mechanism independent of ER-{alpha}. Ovariectomy tended to further reduce eNOS protein content in ER{alpha}KO mice (P = 0.15, ER{alpha}KO vs. ER{alpha}KO ovariectomy). eNOS protein was significantly reduced (P < 0.01) in arteries from ovariectomized ER{alpha}KO mice compared with WT (Fig. 5A). Ovariectomized WT mice were treated with pharmacological levels of E2, but this elevation of E2 did not alter eNOS protein content. In ovariectomized ER{alpha}KO mice, pharmacological estrogen replacement (Fig. 5A) restored eNOS protein to levels similar to those seen in intact ER{alpha}KO and WT mice; however, in ovariecomized WT mice, administration of the same dose of estrogen resulted in significantly higher eNOS protein levels than those seen in ovariectomized ER{alpha}KO mice after estrogen treatment. Disruption of ER-{alpha} also tended to reduce eNOS protein of aortic segments. Ovariectomy of ER{alpha}KO mice resulted in a slight decrease in eNOS expression (P = 0.14, ER{alpha}KO vs. ER{alpha}KO ovariectomy) in aortic segments.



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  		Fig. 4. Effect of NG-nitro-L-arginine methyl ester (L-NAME) on vasodilation to acetylcholine in coronary arteries from WT (A) and ER{alpha}KO (B) mice.

 


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  		Fig. 5. A: endothelial nitric oxide (NO) synthase (eNOS) protein content in coronary arteries from WT (WT; n = 13), ER{alpha}KO (KO; n = 13), ovariectomized ER{alpha}KO (KO OVEX; n = 9), ovariectomized WT mice treated with E2 (WT OVEX + E2; n = 9), and ovariectomized ER{alpha}KO mice treated with E2 (KO OVEX + E2; n = 18). Representative immunoblots are shown above the graph. Each lane was loaded with 1 or 2 µg total protein pooled from arteries from 2–3 mice. Values are expressed relative to WT mean from the same gel. B: eNOS protein content in aorta from WT (n = 3), ER{alpha}KO (n = 3), ovariectomized ER{alpha}KO (n = 9), and ovariectomixed ER{alpha}KO mice treated with E2 (n = 9). Values are means ± SE. *P <= 0.05 vs. WT; #P <= 0.05 vs. WT OVEX + E2.

 

SOD-1 expression. SOD-1 protein levels were increased in coronary arteries of ER{alpha}KO mice compared with levels in coronary arteries of WT mice (Fig. 6). In coronary arteries of WT pharmacological E2 supplementation (800 pg/ml) caused an increase in SOD-1 protein content (P = 0.08, WT vs. WT ovariectomy + E2). In ER{alpha}KO mice, ovariectomy resulted in a significant decrease in SOD-1 protein content (Fig. 6). The E2 implant restored SOD-1 protein content to levels measured in coronary arteries from intact WT and ER{alpha}KO mice.



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  		Fig. 6. Cu/Zn superoxide dismutase (SOD-1) protein content in coronary arteries from WT (n = 13), ER{alpha}KO (KO; n = 13), ovariectomized ER{alpha}KO (KO OVEX; n = 9), ovariectomized WT mice treated with E2 (WT OVEX + E2; n = 9), and ovariectomized ER{alpha}KO mice treated with E2 (KO OVEX + E2; n = 18). Representative immunoblots are shown above the graph. Each lane was loaded with 1 or 2 µg total protein pooled from arteries from 2–3 mice. Values are expressed relative to WT mean from the same gel. *P <= 0.05 vs. WT; +P <= 0.05 vs. KO; #P <= 0.05 vs. WT OVEX + E2; and ++P <= 0.05 vs. KO OVEX.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The purpose of this study was to determine the role of ER-{alpha} in mediating the effects of estrogen on NO-mediated vasodilation of mouse coronary arteries. The main findings of this study are the following: 1) NO-mediated vasodilation is preserved in coronary arteries from ER{alpha}KO mice, 2) SOD-1 protein content increased in coronary arteries from ER{alpha}KO mice, 3) ovariectomy reduced NO-mediated vasodilation and protein levels for eNOS and SOD-1 in ER{alpha}KO mice, and 4) E2 supplementation restored NO-mediated vasodilation and protein content of eNOS and SOD-1 in ovariectomized ER{alpha}KO mice. Thus NO-mediated dilation is preserved in ER{alpha}KO mice through compensatory activation of ER-{alpha} independent pathways.

Long-term estrogen treatment is known to increase vasodilator responses of isolated coronary arteries. In guinea pigs (34) and monkeys (37, 38), chronic estradiol treatment enhances endothelium-dependent vasodilation to acetylcholine. Twenty-four hours of exposure to E2 enhanced relaxation of porcine coronary rings to the calcium ionophore A-23187 (2). Two subtypes of the estrogen receptor, ER-{alpha} and ER-{beta}, have been identified in coronary arteries (16, 27). Estrogen influences gene expression in vascular tissue by activation of one or both of these estrogen receptors. Both ER-{alpha} and ER-{beta} are expressed in vascular endothelial and smooth muscle cells (16, 20, 24, 27, 28). In the present study, ovariectomy caused only a slight, nonsignificant decrease in acetylcholine-induced vasodilation of coronary arteries of WT mice. In contrast, ovariectomy significantly decreased acetylcholine-induced vasodilation in ER{alpha}KO mice and E2 supplementation restored the response. These results indicate that estrogen modulates NO-mediated dilation of coronary arteries through an ER-{alpha} independent pathway. Other reports (13, 40) have indicated that E2 can exert effects on cardiovascular tissue through mechanism(s) independent of ER-{alpha}. Iafrati et al. (13) demonstrated that physiological levels of E2 produced similar inhibition of the vascular response to injury in carotid arteries of ER{alpha}KO and WT mice. Zhu et al. (40) recently reported that estrogen attenuates constrictor responses of mouse aorta by an ER-{beta}-mediated increase in expression of inducible NOS. Estrogen treatment enhanced myogenic constriction of denuded cerebral arteries from ER{alpha}KO but not from WT mice, suggesting the presence of an estrogen-sensitive pathway in vascular smooth muscle lacking ER-{alpha} (8). Although results of the present study do not directly implicate a role for ER-{beta} in modulation of NO-mediated vasodilation of coronary arteries, they do provide evidence that an ER-{alpha} independent pathway regulates endothelial function in these arteries. Activation of this pathway appears sufficient to preserve acetylcholine-induced vasodilation despite the absence of ER-{alpha}. Interestingly, our data suggest that in the absence of ER-{alpha}, this novel estrogen pathway becomes a critical regulator of NO-mediated vasodilation because ovariectomy resulted in significant impairment of acetylcholine-induced vasodilation in ER{alpha}KO mice and the dilation was restored on supplementation with E2.

eNOS protein content was slightly reduced in coronary arteries and aorta from ER{alpha}KO mice compared with WT mice. These findings are consistent with previous reports (11, 17, 22, 35) indicating that estrogen modulates eNOS expression and activity. Aortic endothelial cell NO production was correlated with estrogen receptor expression in WT mice (28), and E2 has been shown to enhance the activity of eNOS in human umbilical vein cells (11) and cultured human uterine arteries (26). In ER{alpha}KO mice, coronary nitrite production during reperfusion after ischemia was reduced compared with that of WT mice (39). Geary and colleagues (8) recently reported that estrogen treatment produced greater endothelium-dependent vasodilator activity and upregulated eNOS protein in cerebral vessels from WT but not ER{alpha}KO mice. Our results indicate that estrogen treatment increased eNOS protein in coronary arteries of ovariectomized ER{alpha}KO but did not restore eNOS protein levels to those of ovariectomized WT mice treated with the same dose of estrogen (Fig. 5). Our results indicate that estrogen modulates eNOS protein expression in coronary arteries through action on ER-{alpha} and through an ER-{alpha} independent pathway.

Other evidence in the literature also indicates that estrogen influences NO production in the endothelium through activation of ER-{alpha} (8, 39). Although our results indicate that estrogen modulates arterial eNOS expression through an ER-{alpha} pathway, our results also demonstrate that neither ovariectomy nor E2 treatment altered vasodilator responses to acetylcholine in WT mice. Preservation of this vasodilator response in ovariectomized mice may be due to input from a compensatory mechanism(s) activated in the absence of estrogen. Upregulation of endothelial prostanoids has been reported in skeletal muscle arterioles and cerebral arteries from ovariectomized rats and mice (7, 12). In the present study, neither disruption of ER-{alpha} nor ovariectomy independently resulted in significant impairment of acetylcholine-induced vasodilation (Figs. 1 and 2B). Compensatory endothelial pathways, for example prostanoid- or EDHF-mediated dilation, may be stimulated by ovariectomy and thus act to maintain endothelium-dependent vasodilator responses of coronary arteries. However, in the absence of both ER-{alpha} and estrogen regulation (Fig. 2C), compensatory mechanisms were absent as indicated by significant impairment of endothelium-dependent vasodilation in coronary arteries in ovariectomized ER{alpha}KO mice.

In coronary arteries of ER{alpha}KO mice, SOD-1 protein expression was increased compared with WT mice. Ovariectomy significantly decreased SOD-1 protein content in coronary arteries of ER{alpha}KO mice, and E2 treatment reversed the effect of ovariectomy, further indication that estrogen regulation of vascular protein expression can occur independent of ER-{alpha}. SOD-1 scavenges superoxide anion and reduces the formation of peroxynitrite from its reaction with NO. This, in turn, preserves the availability of NO as a vasodilatory agent in vascular smooth muscle. Other investigators (1, 6) have reported that sex hormones modulate antioxidant activity in vascular tissue. Young males demonstrate greater oxidative stress than premenopausal females (14). Estrogen reduces lipid peroxidation (29, 32) and has been shown to reduce the production of superoxide anions and increase the biological reactivity of NO in bovine aortic endothelial cells (1). Our results indicate that estrogen activation of an ER-{alpha} independent pathway can stimulate SOD-1 expression and thus compensate for the loss of eNOS protein expression in ER{alpha}KO mice by increasing the bioavailability of NO. Our results suggest that disruption of ER-{alpha} stimulates SOD-1 protein expression in ER{alpha}KO mice; however, this stimulation of SOD-1 expression may only occur as a compensatory response in the presence of high levels of circulating estrogen. Although our results suggest that estrogen modulation of SOD-1 expression occurs through an ER-{alpha} independent pathway, we did not determine whether physiological levels of estrogen regulate SOD-1 expression in WT mice.

Reports (3, 8, 24, 36, 40) in the literature indicate that estrogen also affects smooth muscle vasoreactivity; however, the present data indicate that estrogen modulates endothelium-mediated vasodilation of coronary arteries through an effect on production and/or release of NO by the endothelium. Responses to sodium nitroprusside were not altered in coronary arteries of ER{alpha}KO mice and were not affected by ovariectomy in either WT or ER{alpha}KO mice. Thus neither disruption of ER-{alpha} nor removal of circulating estrogen altered vascular smooth muscle responsiveness to exogenous NO.

In conclusion, the results of this study demonstrate modulation of eNOS protein content of mouse coronary arteries through an ER-{alpha} dependent and an ER-{alpha} independent mechanism; however, endothelium-dependent, NO-mediated vasodilation of coronary arteries can be maintained after disruption of ER-{alpha}. In mice lacking ER-{alpha}, ovariectomy reduces endothelium-dependent, NO-mediated vasodilation. ER-{alpha}-independent upregulation of SOD-1 protein levels may provide an alternate mechanism for maintenance of the biological activity of NO and endothelium-dependent, NO-mediated vasodilation in coronary arteries of ER{alpha}KO mice. Further study is needed to determine whether modulation of endothelium-dependent, NO-mediated vasodilation in coronary arteries occurs through an ER-{beta} pathway.


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant PO-1-HL-52490 (to M. H. Laughlin) and American Heart Association Established Investigator Award 9640177N (to E. M. Price).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the expert technical assistance provided by Pam Thorne and Leslie Newton.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Muller-Delp, Dept. of Health and Kinesiology, Texas A&M Univ., College Station, TX 77843 (E-mail: jmd{at}hlkn.tamu.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.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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 REFERENCES
 

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