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Originally published In Press as doi:10.1074/jbc.M501244200 on March 11, 2005

J. Biol. Chem., Vol. 280, Issue 20, 19704-19710, May 20, 2005
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Estrogen Induces Vascular Wall Dilation

MEDIATION THROUGH KINASE SIGNALING TO NITRIC OXIDE AND ESTROGEN RECEPTORS {alpha} AND {beta}*

Xiaomei Guo{ddagger}, Mahnaz Razandi§||, Ali Pedram§||, Ghassan Kassab{ddagger}, and Ellis R. Levin§||

From the ||Division of Endocrinology, Veterans Affairs Medical Center, Long Beach, California 90822 and the Departments of §Medicine and {ddagger}Biomedical Engineering, University of California, Irvine, California 92717

Received for publication, February 2, 2005 , and in revised form, March 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen has been shown to affect vascular cell and arterial function in vitro and in vivo. Here we examined the ability of estradiol (E2) to cause rapid arterial dilation of elastic and muscular arteries in vivo and the mechanisms involved. E2 administration caused a rapid increase in the outer wall diameter of both types of arteries in ovariectomized female mice. This resulted from estrogen receptor (ER)-mediated stimulation of nitric oxide production, demonstrated by preinjecting the mice arteries with a soluble inhibitor of nitric oxide (monomethyl L-arginine) and by showing the absence of E2 action in eNOS-/- mice. Rapid activation of both ERK/MAP kinase and phosphatidylinositol 3-kinase activity was found in the E2-exposed arteries, and inhibiting either kinase prevented the vasodilatory action of E2. Kinase activation and vasodilator responses to E2 were absent in either ER{alpha} or ER{beta} knock-out mice, implicating both receptor subtypes as mediating this E2 action. These results indicate that E2 modulation of arterial tonus through plasma membrane ER and rapid signaling could underlie many previously observed actions of estrogen reported to occur in women.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen has been suggested to modulate vascular physiology and function from a variety of studies in cellular, animal, and human models. Administration of sublingual estradiol to women significantly reduces exercise-induced myocardial ischemia (1). This perhaps is related to estradiol (E2)1 binding the estrogen receptor (ER) and stimulating nitric oxide (NO) production (2). Genetic deletion of ER{beta} results in the development of hypertension in middle aged female and male mice (3), possibly from the loss of E2-induced NO, resulting in endothelial dysfunction and oxidative stress (4). Relevant to this proposed model, E2 rescues rodents from ischemia-reperfusion injury of their small arteries in muscle, via signaling to NO (5). E2 decreases myocardial infarct size, prevents ventricular arrhythmia, and preserves cell structure in ischemia-reperfusion injury of the heart in animal models (6). E2 also decreases myocardial infarct size and prevents ventricular arrhythmia in women (7, 8). In some instances these effects of E2 are nongenomic, hypothetically resulting from rapid signaling by E2 (9). In contrast, conjugated estrogen plus medroxyprogesterone does not prevent the occurrence of primary or secondary arteriosclerotic heart disease (10, 11). This indicates that estrogen may modulate discrete aspects of cardiovascular tone and function, unrelated to the pathogenesis of atherosclerosis.

One potentially important action of estrogen is to induce arterial dilation. Vasodilation opposes excessive vasoconstriction that results in decreased blood flow and increased systemic vascular resistance. How might E2 rapidly induce dilation of the arterial wall? This is likely to occur through binding vascular cell receptors, because ER{alpha} and ER{beta} are present in vascular endothelial and smooth muscle cells (12, 13). Traditionally, it is felt that steroid sex hormones act in cells after binding their nuclear receptors (14). The steroid-receptor complex then binds specific response elements in the 5' promoter region of target genes. Gene transactivation may also result from more complex protein-protein interactions between the ER, transcription factors such as AP-1, co-activators such as SRC-1, and the basal transcriptional machinery proteins.

Additionally, there is evidence for plasma membrane-localized ER that rapidly signal through G proteins to discrete cell biological functions (reviewed in Ref. 15). In vitro, E2 rapidly stimulates the release of prolactin (16), cAMP (17), and triggers a calcium spike in seconds (18). E2 activates signal cascades that culminate in the activation of ERK, a member of the MAP kinase family (19). E2 preserves neurons from in vitro insults that cause apoptosis or necrosis via signaling through ERK activation (20). Also, E2 rapidly activates NO synthase activity via ERK or PI3K and membrane ER in endothelial cells (EC) (21, 22). The sex steroid prevents the vascular smooth muscle response in rodents to acute vascular injury (angioplasty), preventing the closure of the vessel lumen (23). We showed previously in vitro that E2 stimulates a p38 MAP kinase-MAPKAP-2 and heat shock protein 27 cascade, leading to EC survival and the formation of primitive capillary tubes (9).

Here we investigated the ability of intra-arterial E2 and its mechanism to effect rapid vascular dilation. We found that the sex steroid acts in both elastic and muscular arteries and causes vasodilation through rapid signal transduction to NO generation. These effects were mediated by both the ER{alpha} and ER{beta} receptors and were likely the result of membrane ER action.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Preparation—Homozygous inbred mice (C57BL/10 strain) with body weight of 22.8 ± 2.9 g (mean ± S.D.) and age 80.6 ± 9.7 days were used in this study. Thirty-eight mice underwent ovariectomy by the vendor, and the remaining 14 mice (6 females and 8 males) were intact for comparison. In additional studies, ovariectomized ER{alpha} genedeleted mice, ovariectomized ER{beta} KO mice, and ovariectomized eNOS KO mice were used (n = 4–6 mice/group). Also, 8 wild type mice (same age and strain) were used for comparison to the KO mice. The mice were preanesthetized with intraperitoneal injections of ketamine (56.2 mg/kg) and midazolam (3.75 mg/kg) to achieve requisite immobilization and then were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg). Carotid blood pressure was measured by inserting a catheter into the common carotid artery, and femoral artery pressure was measured through insertion of a catheter into the femoral artery connected to a pressure transducer. 200 units/ml heparin was administered to prevent blood clots via a jugular vein catheter. All animal experiments were performed in accordance with national and local ethical guidelines, including the Institute of Laboratory Animal Research Guide, Public Health Service policy, Animal Welfare Act, and University of California, Irvine, policies regarding the use of animals in research.



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  		FIG. 1.
E2 activates rapid arterial dilation. Ratio of the effects of E2 and Krebs solution on carotid (A) and femoral (B) artery wall diameter in mice. Arteries were filled with Krebs solution or 10 nM E2 in Krebs solution, and arterial wall diameter was measured across a range of hydrostatic pressures. In some cases, ICI192780 was administered alone or prior to E2. Data are mean ± S.E., analyzed by analysis of variance plus Schefe's test. *, p < 0.05 for E2 versus ICI182780 or E2 + ICI182780 in femoral, and E2, ICI182780, or both versus Krebs in carotid artery (n = 6/group).

 
Vasodilatory Effects of E2—In the first experiment, the cannulated femoral artery was loosely ligated in the proximal (cephalad) position, and the artery was then filled with Krebs-Ringer lactate (KRL) solution. Blood pressure measurements were continuously recorded over a 10-min period. On this stable background, the ligature was released. The artery was re-ligated, and the vessel was filled with E2 at 0.01, 0.1, 1, or 10 nM in KRL solution, and the pressure was varied between 60 and 150 mm Hg, in increments of 30 mm Hg. The same study was later repeated in the carotid artery (order reversed in alternating mice). To determine whether the effect of E2 was mediated through ER action, 10-6 M ICI182780, a potent and specific ER antagonist, was dissolved in KRL and used to fill the ligated carotid and femoral arteries for 10 min, and the distension protocol and the relevant parameters were recorded. At that time, the volume was withdrawn, and the artery was re-ligated. The segment was then filled with 10 nM E2 + ICI182780 in KRL, and the parameters were recorded for 20 additional min. Pressure diameter relations and the response to E2 in the ovariectomized and intact mice were recorded. The external geometry of the artery at a given pressurized state was photographed to obtain the loaded outer diameter and the in vivo axial length (using the Aver TV 4.0 program). The in vivo outer diameter was quantified from the images, using a morphometric analysis system (SigmaScan version 5.0). Data were analyzed by two-way analysis of variance, p < 0.05, as significant for comparisons of control versus E2.

The Role of NO—In the next set of experiments, we examined if the rapid vasodilation induced by E2/ER was because of the release of an endothelial-derived relaxing factor substance, most likely NO. The arteries were first filled for 10 min with KRL and then1 µM monomethyl L-arginine (L-NAME), a nitric-oxide synthase inhibitor in KRL, then filled with 10 nM E2 + L-NAME in KRL for 20 min. To implicate further NO, the effect of E2 alone was assessed in ovariectomized eNOS-/- mice (The Jackson Laboratories). To determine the signaling required for E2/ER to release NO in vivo, the arteries were filled with PD98059 (1 µM), a specific MEK inhibitor that was previously shown to block E2-induced ERK activation in vitro. This was compared with the ligated artery filled with 10 nM E2 + PD98059. To determine a contribution from PI3K activation, the arteries were filled with a PI3K inhibitor, wortmannin (100 µM), for 10 min of recording, prior to exposing the artery to 10 nM E2 + wortmannin.

ER KO Studies—We then determined which ER mediates the ability of E2 to induce vasodilation. Ovariectomized ER{alpha} KO mice were created by Lubahn et al. (24), and ER{beta} KO mice were produced by the Wyeth Research Institute (25). The femoral and carotid arteries were utilized as described above, and the data shown are at 120 mm Hg.

In Vivo Kinase Activity—The carotid or femoral artery was filled with 10 nM E2, or KRL alone as control, for the 10 min of the experiment. The isolated artery segment was rapidly excised, with the distal and proximal segments suture ligated, and the mouse was rapidly euthanized. The artery segments were dropped into liquid nitrogen, pulverized, and solubilized in kinase binding buffer, and ERK or AKT protein was immunoprecipitated. Kinase activity was determined in vitro as described previously (26). Myelin basic protein was used as substrate for ERK activity, and an antibody to phosphorylated AKT (Ser-473) reflected PI3K activity, as determined by Western blot. ERK or AKT total protein was shown to normalize activity results. The activity was derived from 3 to 5 pooled arteries from separate mice



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  		FIG. 2.
Nitric oxide mediates E2 and ICI182780-induced vasodilation. A, ICI182780-induced dilation in carotid artery. *, p < 0.05 for ICI182780 versus L-NAME or ICI182780 +L-NAME. B, E2 action in either artery is prevented by L-NAME. L-NAME was administered alone or 10 min prior to ICI182780 or E2. *, p < 0.05 for E2 versus L-NAME or L-NAME + E2 by analysis of variance. C, lack of E2-induced dilation in eNOS-/- mice (n = 5/group).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
E2 Rapidly Dilates Elastic and Muscular Arteries—Female ovariectomized mice were anesthetized, and their carotid (elastic) and femoral (muscular) arteries were sequentially cannulated, allowing for sensitive measurements of arterial wall diameter, reflecting constriction or dilation. Each isolated artery was filled with Krebs-Ringer lactate (KRL) solution, and the arterial wall outer diameter was measured optically at various imposed static pressures (60–150 mm Hg), each over 20 min. These measurements were compared with arteries filled with KRL containing various concentrations of E2. The sex steroid produced a significant increase in arterial wall diameter in both arteries within 2 min, compared with KRL alone, and was seen at all pressures (Fig. 1A). This occurred at concentrations as low as 1 nM E2 (data not shown).

To determine whether this was mediated through ER action, the arteries were first filled for 10 min with KRL and 1 µM ICI182780, a potent and specific ER antagonist. ICI182780 specifically blocked vasodilation induced by 10 nM E2 but only in the femoral artery (Fig. 1B). Most interestingly, ICI182780 by itself acted as an agonist in the carotid artery, inducing dilation comparable with E2 through a similar mechanism (Fig. 1A and see below). Intact female or male mice did not respond to E2, probably because of the effects of endogenous E2 (data not shown).

NO Mediates the Vasodilator Effect of E2—A possible mediator of the vasorelaxing action of E2 is NO. We initially implicated this vasodilator, in that E2-induced arterial dilation in either artery was substantially prevented by brief pretreatment with the nitric-oxide synthase inhibitor, monomethyl L-arginine (L-NAME) (Fig. 2B). Most interestingly, the novel agonist effect of ICI182780 in the carotid artery was also prevented by L-NAME (Fig. 2A).



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  		FIG. 3.
E2 or ICI182780 signals to vasorelaxation via kinases. E2-induced arterial dilation is prevented by ERK/MAP kinase inhibition (PD98059 (PD)) (A) and PI3K inhibition (wortmannin (wort)) (B). Arteries were filled with Krebs, then PD98059 (PD) or wortmannin (Wort) in Krebs, and then E2 + either kinase inhibitor. *, p < 0.05 for E2 versus PD98059, or E2 + PD98059, or E2 versus wortmannin, wortmannin + E2 (n = 6). C, lack of vasodilation by ICI182780 (ICI) in carotid arteries pre-exposed to PD98059 (PD) or wortmannin (n = 5). +, p < 0.05 for ICI182780 versus ICI182780 + PD98059 or ICI182780 + wortmannin (Wort).

 



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  		FIG. 4.
Estrogen or ICI182780 activates ERK and PI3K in vivo. A, arteries were exposed to E2 ± PD98059, or wortmannin (Wort), or kinase inhibitor alone. The arteries were excised, and kinase activity was determined as described under "Experimental Procedures." A representative study and a bar graph of three experiments combined is shown. *, p < 0.05 for control versus E2; +, p < 0.05 for E2 versus E2 + kinase inhibitor, p < 0.05. B, ICI182780 (ICI) stimulates PI3K and ERK activity in the carotid artery. The study shown represents kinase activity from the pooled carotid arteries of five ovariectomized female mice. MBP, myelin basic protein. PD, PD9805.

 
To implicate further NO, we determined the effect of E2 in ovariectomized eNOS-/- mice. The rapid arterial dilation induced by E2 in ovariectomized wild type mice was not seen in ovariectomized eNOS-deleted mice (Fig. 2C). Here the results at physiological pressure (120 mm Hg) are shown and do not vary significantly over a range of 60–150 mm Hg. Most interestingly, the basal diameter of the artery was decreased in the eNOS KO mice, compared with wild type mice. This indicates an important contribution of NO to basal arterial tone (Fig. 2C and data not shown). These results indicate that acute arterial wall vasodilation occurs in response to E2, mediated through a rapid cross-talk from ER to eNOS, with subsequent NO production and action.

E2 Stimulates Rapid Kinase-induced Arterial Dilation—Our results suggested a possible mechanism whereby nongenomic, rapid signaling from membrane ER up-regulates NO synthase enzymatic activity. Subsequent NO production then mediates the vasodilation action of the sex steroid, as shown above. In cultured cells, E2 has been reported to signal rapidly through ERK/MAP kinase and PI3K to induce NO synthase activity and NO generation (21, 22). This is likely to occur through membrane ER, because ER targeted to the plasma membrane of cells supports kinase activation by E2. In contrast, ER targeted to the cell nucleus does not support rapid activation of ERK (27).

We then determined the role of rapid signaling pathways mediating this E2 action in vivo. We first showed that brief pretreatment of the arteries with soluble inhibitors of ERK (PD98059) or PI3K (wortmannin) each prevented E2-induced arterial dilation. This occurred in both arteries over the entire range of pressures (Fig. 3, A and B). Most interestingly, inhibition of either kinase also prevented the vasodilator effect of ICI182780 in the carotid artery (Fig. 3C).



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  		FIG. 5.
E2 or ICI182780 has no vasodilatory effect in ER KO mice. A, cannulated arteries of wild type (WT) or ER{alpha} or ER{beta} KO mice were exposed sequentially to Krebs solution or E2, and the diameter ratio was calculated (n = 6/group). *, p < 0.05 for wild type versus KO in carotid artery; +, p < 0.05 for same in femoral artery. B, carotid arteries of wild type, ER{alpha}, or ER{beta} KO mice were cannulated then exposed to IC182780 (ICI) (n = 5 for each mouse group); *, p < 0.05 for Krebs versus ICI182780. C, lack of kinase activation in the carotid arteries of KO mice. PI3K and ERK activity in response to either E2 or ICI182780 (ICI) was determined in the carotid arteries pooled from four ER{alpha} and four ER{beta} KO mice.

 
To support further the roles of these signal molecules in E2 action, we determined whether E2 activated these kinases in vivo. We measured arterial wall ERK and PI3K activity after exposing the lumen of the whole arterial segment in the anesthetized mouse to 10 min of 10 nM E2. E2 induced significant increases in ERK activity, and AKT phosphorylation at serine 473 (the latter reflecting PI3K activity) (Fig. 4A). Similarly, ICI182780 stimulated ERK and PI3K activities in the carotid artery (Fig. 4B) but not in the femoral artery (data not shown). Thus, we propose that rapid stimulation of these kinases by E2 induces the up-regulation of eNOS activity. This leads to the increased production of NO and subsequent vascular wall relaxation. A similar mechanism underlies the agonist effects of ICI182780 in the carotid artery.



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  		FIG. 6.
Schematic of E2 vasodilator effects in the artery. EC, endothelial cell; VSMCs, vascular smooth muscle cell.

 
ER{alpha} and ER{beta} Mediate the Vasodilator Effect of E2—Which ER mediates the effects of E2? This is an important issue because there is evidence that ER{alpha} or ER{beta} underlies the various effects of E2 in the vasculature (3, 23). To determine this, we carried out studies in ovariectomized female ER{alpha} and ER{beta} KO mice. The KO mice were created as described previously (24, 25). E2 had no significant effect in either the carotid or femoral arterial beds of either the ER{alpha} or ER{beta} KO mice. In contrast, E2 caused dilation of the arteries of wild type mice (Fig. 5A). ICI182780 also caused dilation in the carotid artery of wild type mice, not seen in either KO mouse (Fig. 5B). Failure to dilate in these mice correlated with the inability of E2 or ICI182780 to stimulate ERK or PI3K activity in the carotid arteries from ER{alpha} or ER{beta} KO mice (Fig. 5C). Thus, it is E2 signaling to kinase activation via either receptor that mediates the acute dilation effects of the sex steroid in arteries. This is consistent with in vitro studies that implicate actions of membrane ER{alpha} or ER{beta} to up-regulate eNOS activity (28).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results define a novel mechanism by which estrogen modulates in vivo vascular tone. This suggests that E2 might mitigate endogenous mechanisms that increase vascular wall tone and could be important in the absence of estrogen input as occurs after the menopause. In women with coronary artery disease, E2 administration attenuates the paradoxical acetylcholine-induced coronary constriction (29). Administration of premarin plus medroxyprogesterone to post-menopausal women for 3 months results in a significant increase in forearm blood flow, reflecting vasodilation (30). In healthy young women, flow-mediated and NO-mediated arterial compliance of the forearm significantly changes during the menstrual cycle, correlating to estrogen blood concentration (31). Additional chronic studies in humans support a mechanistically undefined relationship between estrogen and vasodilation (32, 33).

How does E2 induce an increase in arterial wall diameter? Here we show that eNOS and nitric oxide mechanistically underlie this action of the sex steroid. The NO inhibitor, L-NAME, prevents the effects of E2, implying that eNOS activation and NO production from EC are important. More definitively, we showed that E2 does not produce the demonstrated vascular effects in the eNOS -/- mouse. eNOS is responsible for the majority of NO production from the EC. In vivo studies have shown that basal eNOS production is compromised in ER{alpha}-deleted mice (34). E2 can also stimulate inducible nitricoxide synthase and NO production from vascular smooth muscle cells or arteries that have been denuded of endothelium (3). This may contribute to the observation that middle-aged male and female ER{beta} knock-out mice develop moderately severe hypertension (3). Thus, it may be that in the shorter term, eNOS stimulation from EC predominates, whereas longer term modulation may reflect NO production from both vascular cells through different NOS and ER isoforms.

The ability of E2 to stimulate NO-mediated vasodilation appears to be linked to the stimulation of ERK and PI3K. We found that E2 activated both these kinases in the in vivo arterial segment filled with E2. This action of E2 was blocked by an ER antagonist and by specific soluble inhibitors of MEK (the kinase immediately upstream to ERK) and PI3K. This further correlated with the ability of PD98059 and wortmannin to inhibit the vasodilation induced by E2 in vivo. E2 can modulate EC paracellular permeability through eNOS- and inducible nitric-oxide synthase-related actions in vitro (35). We showed recently that E2 can activate more than 250 genes in a PI3K-dependent fashion, after exposure of EC to this sex steroid for only 40 min (36). This includes genes such as Egr-1 that are induced by acute vascular injury and are proposed to mitigate vascular damage in vivo (37) Thus, the ability of E2 to activate rapid in vivo signaling through kinase cascades is meaningful to preserve beneficial vascular function. This reflects both rapid nongenomic and genomic actions in the vasculature.

E2 rapidly signals to these kinase cascades, and this most likely originates from the small population of ER at the plasma membrane (15, 27). The membrane receptor is likely to be the nuclear receptor, translocated to the membrane (38). In support of this idea, we recently reported that EC prepared from ER{alpha}/ER{beta} double knock-out mice fail to show detectable ER at the membrane or in the nucleus (39). This suggests that the two receptor pools (membrane and nucleus) are products of the same ER genes coding for the {alpha} and {beta} isoforms, respectively. The membrane population is responsible for rapid signal transduction through multiple pathways including those identified here. This idea is further supported in that targeted expression of ER{alpha} in the nucleus of ER-negative cells fails to support E2-induced rapid signaling, whereas expression at the membrane supports these functions (27). It is ER at the membrane, for instance, that physically associates with the p85 regulatory subunit of PI3K in cells (5) and activates NO (28). In cells, the integrated functions of membrane and nuclear ER result in the overall ability of E2 to modulate biology.

As a novel action, we report that ICI182780 rapidly stimulates vasodilation via ERK and PI3K signaling, and eNOS activation, similarly to E2. This only occurs in the carotid artery and not the femoral artery. In the latter, ICI182780 antagonized the effects of E2. The mechanism by which ICI182780 antagonizes the established rapid signaling from membrane ER (40) has not been determined. This may be related to ICI182780 promoting a rapid internalization of membrane ER to endosomes (41) or hastening receptor degradation through undetermined mechanisms. We speculate that in the carotid artery, ICI182780 does not enact the antagonistic mechanism found in the femoral artery. Furthermore, the conformation of the membrane ER in the carotid artery may allow ICI182780 to engage the receptor as an agonist. Further studies will be needed to dissect the differential mechanisms, using both in vitro and in vivo models.

We also found that both ER{alpha} and ER{beta} mediate the actions of E2 shown here (Fig. 6). This was based on the lack of E2-induced dilation in the arteries of ER{alpha} or ER{beta} KO mice. Previous studies have indicated that both receptors are present on EC (28, 42). We recently showed that at the membrane of endothelial cells, ER{alpha} and ER{beta} exist both as homodimers and as heterodimers (39). Karas and co-workers (23) show that it is ER{alpha} that prevents the smooth muscle/medial arterial response to balloon angioplasty in rodents (23). In a more chronic model, ER{beta} plays an important role in the prevention of hypertension (3). Thus, both ER isoforms are relevant to the modulation of vascular function and, as shown here, in vivo vasodilation. ER{alpha} receptors are present in ER{beta} KO mice blood vessels, and the reverse is also true. We therefore propose that it is the disruption of the functional heterodimer by either receptor deletion that results in the loss of E2 action. Overall, rapid signaling as a mechanism of estrogen action at the membrane may contribute to the beneficial effects of arterial relaxation, potentially preventing the pathological effects of excessive vasoconstriction (43).


   FOOTNOTES
 
* This work was supported by grants from the Research Service of the Department of Veterans Affairs and National Institutes of Health Grants HL59890 (to E. R. L.) and HL055554-06 (to G. K.), and American Heart Association Grant 0140036N (GSK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Medical Service (111-I), Long Beach Veterans Affairs Medical Center/University of California, 5901 E. 7th St., Long Beach, CA 90822. Tel.: 562-826-5748; Fax: 562-826-5515; E-mail: ellis.levin{at}med.va.gov.

1 The abbreviations used are: E2, estradiol; ER, estrogen receptor; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; KO, knockout; L-NAME, monomethyl L-arginine; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MEK, MAP kinase/ERK kinase; PI3K, phosphatidylinositol 3-kinase; ER, estrogen receptor; EC, endothelial cell. Back


   ACKNOWLEDGMENTS
 
We thank Wyeth Research and Heather Harris for supplying the ER knock-out mice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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