Published online before print December 11, 2003, doi: 10.1161/01.RES.0000111525.96232.46
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© 2004 American Heart Association, Inc.
Integrative Physiology |
Estrogen Elicits Cytochrome P450—Mediated Flow-Induced Dilation of Arterioles in NO Deficiency
Role of PI3K-Akt Phosphorylation in Genomic Regulation
From the Departments of Physiology (A.H., D.S., Z.W., C.Y., G.K.) and Pharmacology (M.A.C., H.J.), New York Medical College, Valhalla, NY; and the Department of Biochemistry (J.R.F.), University of Texas, Southwestern Medical Center, Dallas, Tex.
Correspondence to An Huang, MD, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595. E-mail an_huang{at}nymc.edu
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Abstract |
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This study investigated the mechanisms responsible for the estrogen-dependent, cytochrome P450 (CYP)-mediated dilator responses to shear stress in arterioles of NO-deficient female rats and mice. Flow-induced dilation (FID) was assessed in isolated arterioles from NG-nitro-L-arginine methyl ester (L-NAME)-treated male and ovariectomized female rats before and after overnight incubation with 17ß-estradiol (17ß-E2, 10-9 mol/L). In control conditions, prostaglandins (PGs) mediated FID, because indomethacin (INDO) abolished the responses. After incubation of the vessels with 17ß-E2, the basal tone of arterioles was significantly reduced and FID was augmented. INDO did not affect the dilation of the vessels incubated with 17ß-E2. Dilations of these vessels, however, were eliminated by PPOH and miconazole, inhibitors of CYP/epoxygenase. Simultaneous incubation of the vessels with 17ß-E2 plus ICI, 182,780, an estrogen receptor antagonist, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation or the transcriptional inhibitor DRB, prevented the reduced arteriolar tone and the enhanced CYP-mediated FID caused by incubation of vessels with17ß-E2. Western blot analysis indicated a significantly increased phospho-Akt level in arterioles incubated with 17ß-E2 compared with those without 17ß-E2. The enhanced phospho-Akt in response to 17ß-E2 was localized, by immunohistochemistry, to arteriolar endothelial cells. Moreover, GC-MS analysis indicated a significantly increased production of epoxyeicosatrienoic acids, vasodilator metabolites of CYP/epoxygenase, in arterioles incubated with 17ß-E2, a response that was prevented by ICI 182780 and wortmannin, respectively. Thus, estrogen, via a receptor-dependent, PI3K/Akt-mediated pathway, transcriptionally upregulates CYP activity, leading to an enhanced arteriolar response to shear stress.
Key Words: estradiol • flow-induced dilation • cytochrome P450 • Akt • transcription
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Introduction |
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Since the first demonstration of the cardiovascular protective effects of female hormones in endothelial nitric oxide synthase knockout (eNOS-KO) mice in 1998,1 evaluation of the role of estrogen in the regulation of cardiovascular function in pathological states in which the bioavailability of NO is impaired, such as hypertension and heart failure, has become important. This would answer the question whether alternative mechanisms, independent of NO, could also be activated by estrogen. Our previous studies, conducted on skeletal muscle arterioles of female eNOS-KO mice2 and female rats treated chronically with a NOS inhibitor,3 demonstrated that estrogen-related protective mechanisms involving endothelial regulation of shear stress were well preserved, as indicated by the maintenance of augmented flow-induced dilation in vessels of females compared with those of males. Interestingly, the enhanced flow-induced dilation in the vessels of females was mediated by epoxyeicosatrienoic acids (EETs), cytochrome P-450 (CYP)/epoxygenase metabolites of arachidonic acid, which caused dilation of vessels via activation of smooth muscle KCa channels and could therefore be considered as endothelium-derived hyperpolarizing factors (EDHF). Additionally, the EETs/EDHF-mediated flow-induced dilation was purely estrogen dependent, as indicated by the fact that ovariectomy (OV) eliminated, but estrogen replacement restored, the responses.4 Although there are a few studies extant that reported a specific contribution by estrogens to EDHF-mediated responses to vasoactive agents,5,6 the underlying mechanisms involved, specifically those in response to shear stress, remain largely unknown.
Another facet of gender differences related to estrogens and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway has attracted considerable attention.7–9 Premenopausal women display a significantly greater staining of Akt in the nuclei of cardiac myocytes than men and postmenopausal women. This gender difference is also observed in transgenic mice with cardiospecific overexpression of IGF-1, a stimulus of the PI3K signaling pathway.10 In vascular endothelial cells, estrogen activates several signaling pathways, including PI3K-Akt.11 It has been demonstrated that interaction of cell membrane-bound estrogen receptors (ERs) with PI3 kinase phosphorylates Akt, followed by phosphorylation of eNOS, resulting in NO release.12,13 Moreover, PI3K/Akt recruitment attributed to cell membrane-initiated signaling can also affect gene transcription. This was demonstrated by studies showing that exposure of endothelial cells to estradiol for 40 minutes significantly increased the expression of 250 genes, a response that was sensitive to the PI3K inhibition.14 Moreover, regulation of c-fos gene expression by estrogen via activation of a SRE (serum response element) is mediated by PI3K, as well as the mitogen-activated protein kinase (MAPK) pathway.15 Also, a recently published study demonstrating kinase-initiated regulation of transcription factors offers an explanation for the bone-protective effects of estrogen and some of the synthetic sex steroid receptor ligands, whose activity is mediated by extranuclear receptors.16 Thus, the action of estrogen involves rapid activation of kinase cascades, followed by a gene transcription that accounts for estrogen’s bioactivity. A crosstalk between estrogen-stimulated acute increases in cAMP and regulation of gene transcription has also been reported.17
Given that CYP-mediation of flow-induced dilation is dependent on estrogen and that PI3 kinase is targeted by estrogen, we hypothesized that PI3K/Akt phosphorylation plays a key role in signaling pathways involving the estrogen-elicited, CYP-mediation of arteriolar responses to shear stress. The study also aimed to define whether the mechanism by which estrogen regulates the responses is transcriptionally based.
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Materials and Methods |
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Animals
Seven-week-old male and female Wistar rats were purchased from Charles River Laboratories (Wilmington, Mass). Female rats were ovariectomized (OV),4,18,19 and both male and OV rats received NG-nitro-L-arginine methyl ester (L-NAME) in their drinking water (50 mg/100 mL) for 4 weeks. Systolic blood pressure was monitored with a tail-cuff method. All protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the guidelines of the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.
Measurement of Plasma Nitrate/Nitrite (NO2/NO3)
By using a fluorometric assay, plasma concentrations of NO2/NO3 were measured after the plasma was filtered to remove hemoglobin.3,4
Method of Incubation
Experiments were conducted on isolated gracilis muscle arterioles of rats.20 Control vessels and those having been incubated with 17ß-estradiol were isolated from each gracilis muscle of one rat. In E2 incubation experiments, arterioles were incubated overnight (10 to 12 hours) with 17ß-estradiol (17ß-E2, 10-9 mol/L) in a cannulated and pressurized state (50 mm Hg). In separate experiments, time control experiments were performed by incubation of the vessels without estrogen for the same period of time. Sterilized PSS containing 0.1% antibiotic-antimycotic and L-NAME (10-4 mol/L), buffered with 5% CO2, used was not recirculated.
Experimental Protocols
Changes in diameter of arterioles in response to increases in perfusate flow were studied at 80 mm Hg perfusion pressure.
In the first protocol, after control experiments (before incubation), in which flow-induced dilation was determined before and after administration of indomethacin (INDO, 10-5 mol/L), PPOH (3x10-5 mol/L), or miconazole (MCZ, 2x10-6 mol/L) (inhibitors of CYP/epoxygenase), two other vessels isolated from another piece of the muscle of the same rat, were incubated overnight with 17ß-E2 (10-9 mol/L). After incubation, experiments, as those described in control, were repeated.
In this series of experiments, experiments identical to those described in the first protocol were performed by incubating male vessels with 17ß-E2 plus the estrogen receptor (ER) antagonist ICI 182,780 (10-5 mol/L), or 17ß-E2 plus the PI3 kinase inhibitor wortmannin (WTM, 10-7 mol/L), or by incubating vessels of OV rats with 17ß-E2 plus 5,6-dichloro-1ß-D-ribofuranosylbenzimidazole (DRB, 10-5 mol/L), a reversible RNA polymerase II inhibitor.
Western Blotting
Isolated second-order arterioles, 
5 mm in length, were incubated with or without 17ß-E2 (10-9 mol/L) for different times ranging from 30 minutes to 8 hours. Then vessels were solubilized in modified Laemmli buffer and sonicated (2 minutes), before boiling (5 minutes) to denature the proteins.
Samples were loaded on a SDS-PAGE gel. Membranes were probed with primary antibody (polyclonal antibodies of phospho-Akt and Akt) (Cell Signaling), dissolved in PBS with 1% nonfat dry milk and 0.1% tween-20 overnight at 4°C, and then were probed with secondary antibody after washing with PBS. The secondary antibody was conjugated to horseradish peroxidase according to the Amersham ECL-Plus protocol. The exposed film was developed in a Kodak X-Omat developer. Specific bands were normalized to GAPDH.
Immunohistochemistry
After incubation of arterioles with or without 17ß-E2 (10-9 mol/L) for 8 hours, vessels were embedded in OCT compound. Frozen sections (10 µm) were incubated with primary antibody for 24 hours at 4°C. After a wash in PBS, sections were incubated with Cy3-conjugated secondary antibody for 1 hour at room temperature. The fluorescent image was visualized by an Olympus BX60 microscope with a CCD camera.21
Quantitation of EETs
Purification of EETs
Gracilis muscle arterioles, 6 to 8 mm in length (6 to 10 µg protein/per vessel), were isolated and incubated with or without 17ß-E2 (10-9 mol/L), and 17ß-E2 plus ICI 182,780 (10-5 mol/L) or wortmannin (10-7 mol/L), or PPOH (3x10-5 mol/L), respectively, for 8 hours, followed by another 1-hour incubation with NADPH (10-3 mol/L), INDO (3x10-5 mol/L), L-NAME (10-4 mol/L), DDMS (3x10-5 mol/L), and arachidonic acid (10-5 mol/L). Finally, 4.5 ng of a mixture of D8-EETs was added to each sample as internal standards. After extraction, the samples were reconstituted in 20 µL methanol and injected into reverse phase HPLC to obtain EET fractions.2
Derivatization and Quantitation With GC-MS Analysis
After derivatization, the samples were reconstituted in 50 µL iso-octane, and a 10-µL aliquot was injected into a GCMS (HP-5890/5989A, Hewlett-Packard). Endogenous EETs were identified (ion mass-to-charge ratio=319) by comparison of GC retention times with authentic D8-EETs (mass-to-charge ratio=327) standards, quantified by calculating the ratio of abundance and further normalized with protein content.
Chemicals
All chemicals were obtained from Sigma.
Statistics
Changes in diameter in response to increases in flow in each vessel were normalized to passive diameter. Statistical significance was calculated by repeated-measures ANOVA followed by the Tukey-Kramer multiple-comparison test, and Student’s t test. Values are mean±SE. Significance level was taken at P<0.05.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
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Results |
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As a result of L-NAME treatment, plasma concentrations of NO2/NO3 were significantly lower in male (5.95±1.53 µmol/L) and OV (5.51±0.68µmol/L) rats, associated with an elevated systolic blood pressure (180.3±4.7 and 170.8±5.9 mm Hg, respectively), compared with normal rats (P<0.05).3,4 Active (AD) and passive diameters (PD) of arterioles from male and OV rats were comparable in control conditions (AD, 57.9±2.0 versus 56.8±4.1 and PD, 119.3±2.9 versus 115.3±5.7, respectively). As a result, their arteriolar tone was similar. After overnight incubation of arterioles with 17ß-E2, active diameter of both groups of vessels was increased, leading to a significantly attenuated arteriolar tone compared with control (61.1±1.9% versus 48.6±1.2% in males, and 57.0±2.8% versus 48.8±2.0% in OV rats, respectively).
Flow-Induced Dilation
Arteriolar dilations to increases in perfusate flow and the mediators responsible for flow-induced dilation, as a function of incubation with estrogen, are illustrated in Figures 1 (male) and 2 (OV), respectively. In the control condition (top panel), flow-induced dilation was not affected by PPOH, but was sensitive to INDO, indicating a prostaglandin-mediated response. After incubation of the vessels with 17ß-E2 (bottom panel), flow-induced dilation in both groups of vessels was significantly enhanced (by 31% and 36%, respectively), which however, was resistant to INDO and could be abolished by PPOH, revealing that the response is dependent on CYP.
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We tested the specific role of ER in 17ß-E2-induced changes in the mediation of flow-induced dilation. Figure 3, top, shows that the ER antagonist, ICI 182,780, prevented the reduced arteriolar tone and the enhanced PPOH-sensitive flow-induced dilation elicited by incubation with 17ß-E2, as manifested by a response identical to that observed in control conditions (Figure 1, top). To determine whether the PI3K/Akt pathway is involved in the 17ß-E2-induced alterations, the action of estrogen was assessed in the presence of wortmannin in order to inhibit PI3 kinase phosphorylation. Figure 3, middle, shows that similar to ICI 182,780, incubation of vessels with wortmannin in the presence of 17ß-E2 reversed the CYP-mediated dilation elicited by 17ß-E2 alone to the one mediated by prostaglandins, as evidenced by the sensitivity of the response to INDO, similar to the one illustrated in Figure 1, top. To further determine whether gene transcription is required for the effects of 17ß-E2, in separate experiments, arterioles were incubated simultaneously with 17ß-E2 and the transcription inhibitor DRB. As shown in Figure 3, bottom, DRB also prevented the estrogen-elicited, CYP-dependent dilation to shear stress.
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Time control experiments showed that overnight incubation per se did not significantly alter arteriolar responses to flow/shear stress, and moreover, wortmannin had no significant effect on prostaglandin-mediated flow-induced dilation of NO-deficient skeletal muscle arterioles, after 6- to 8-hour treatment of the vessels with the agent (online Figures 1 and 3, respectively, in the online data supplement, available at http://www.circresaha.org).
Molecular Analyses
Evidence provided by Western blot analysis (Figure 4) shows that incubation with 17ß-E2 for 8 hours significantly enhanced phospho-Akt levels in arterioles of L-NAME-treated male rats. We also performed a time-course analysis showing that after exposure of the vessels to 17ß-E2 for 30 minutes, an increase in phospho-Akt level occurred that was inhibited by LY 249003 (10-5 mol/L) (data not shown).
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A specific antibody for phospho-Akt was used to probe cross sections of isolated arterioles for the localization of immunoreactive protein. Online Figure 4 (in the online data supplement available at http://www.circresaha.org.) shows that staining for phospho-Akt occurred in the cytosolic regions of endothelial cells of arterioles incubated with 17ß-E2 (panel a), but was not detected in those incubated without 17ß-E2 (panel c), indicating that endothelial cells are the main source of the estrogen-stimulated production of phospho-Akt in these vessels.
GC-MS Analysis
Figure 5 shows the quantification of EETs by GC-MS analysis in arterioles of L-NAME-treated male rats, indicating that the production of EETs in 17ß-E2-incubated arterioles was significantly enhanced compared with those incubated without estrogen. The 17ß-E2-elicited enhancement of EET production was prevented by wortmannin and ICI 182,780, confirming that the response is PI3K-AKt dependent and is mediated by ERs. PPOH inhibited the release of EETs by 80%.
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Discussion |
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The present study is the first to provide evidence for the role of PI3K-Akt in the mediation of estrogen-dependent, CYP-mediated flow-induced dilation when NO synthesis is absent. It also shows that changes in the mediation of arteriolar responses to shear stress are mediated by estrogen receptors and gene transcription, and that the enhanced production of EETs in response to estrogen is also a PI3K-Akt-dependent response, mediated by activation of estrogen receptors.
Our previous studies demonstrated that estrogen elicits EDHF-mediated flow-induced dilation of skeletal muscle arterioles of rats and mice, in response to NO deficiency.2–4 To the best of our knowledge, there has been no information available with regard to the relationships among estrogen, shear stress and EDHF, albeit a few previous studies did demonstrate effects of estrogen on mediation by EDHF of responses elicited by vasoactive agents,5,6,22 especially in a condition associated with significant impairment of NO synthesis.23 Thus, the present study aimed to extend our previous findings by characterizing the role of intracellular signaling cascades involved, and moreover, by examining whether the effects of estrogen are genomic or nongenomic.
Attenuated Arteriolar Tone After 17ß-E2 Incubation
The basal tone of arterioles of males and OV females was comparable in the control condition, and was significantly attenuated in both groups of vessels after incubation with17ß-E2 (Figures 1 and 2
). Our previous studies, conducted on the same vasculature of male spontaneously hypertensive rats, demonstrated that there was a NO-dependent attenuation of arteriolar tone, after incubation with 17ß-E2, as indicated by elimination of the responses with a NO inhibitor.20 In the present study, NO synthesis has been suppressed before, as well as during, the experiment with L-NAME, evidenced by significantly reduced plasma concentrations of NO2/NO3 and increases in blood pressure. Moreover, inhibitors of the synthesis of endothelial vasodilators, including INDO, PPOH, and MCZ, did not prevent the attenuated basal tone. In this context, we presumed that the attenuated basal tone of arterioles elicited by incubation with 17ß-E2 is not an endothelium-dependent response, but rather a direct effect of estrogen on vascular smooth muscle. The results are in keeping with our own previous studies and those of others showing that 17ß-E2 has a direct vasodilator effect that is independent of the endothelium,24 and which may result from its direct Ca2+ antagonistic effect on smooth muscle via increases or decreases of Ca2+ efflux or influx,25,26 respectively, and also via inhibition of Ca2+ channels27,28 or activation of K+-channels29 of smooth muscle. Alternatively, metabolites of estradiol (during the period of incubation) can also cause vasodilation.30
Endothelial Mediators Contributing to Flow-Induced Dilation in Response to Incubation With 17ß-E2
In the control condition, PPOH did not affect flow-induced dilation, which was eliminated by INDO (top of Figures 1 and 2 and online Figure 1), indicating a prostaglandin-mediated response in arterioles of L-NAME-treated male and OV rats.3,4 Arterioles that had been exposed to 17ß-E2 overnight exhibited a significantly augmented dilation to flow, associated with a response that became INDO-resistant, but was sensitive to PPOH (bottom of Figures 1 and 2). Because overnight incubation of the vessels per se did not affect the magnitude or mediation of flow-induced dilations (online Figure 1), the changes observed (bottom of Figures 1 and 2) are purely due to effects of estrogen. The results showing a switch of the mediator of the response from prostaglandins to CYP metabolites in male phenotypic arterioles, after overnight incubation with 17ß-E2, are identical to those observed previously in arterioles of intact female rats and OV female rats receiving estrogen replacement therapy.3,4 These results support the conclusion that estrogen per se, regardless of whether it is present in vivo or in vitro, is in fact, responsible for the modulation of arteriolar tone and for the enhanced flow-induced dilation via CYP mediation.
The specific mechanisms responsible for the switch of mediators observed in the present experiments are not yet defined. We demonstrated previously that in adapting to the lack of NO, prostaglandins released from both COX-1 and COX-2 isoforms mediate flow-induced dilation in male and OV arterioles,4 perhaps as a result of the absence of the inhibitory effect of NO on COX-2 expression.31 In the presence of estrogen, however, COX-2 mRNA expression is inhibited,32 and moreover, estrogen favors the contribution of EDHF in the mediation of vascular responses.2,3,22,23 Thus, these findings might explain the switch from prostaglandin mediation of the responses in NO deficient male or OV arterioles to one mediated by CYP, when incubated with 17ß-E2.
The mechanotransduction by which endothelial cells convert shear stress to biochemical signals, which are responsible for the release of EETs, have not yet been characterized; especially, the signaling pathway that seems to be recruited specifically by estrogen. However, the role of CYP in vascular dilation/hyperpolarization to shear stress has already been described in coronary arterioles of patients with cardiovascular disorders33 and in skeletal muscle arterioles of rats and mice in the absence of NO syhthesis.2–4 Thus, EETs have been defined as EDHFs on the basis of their effect—in the absence of NO and prostaglandin synthesis—on smooth muscle hyperpolarization in a variety of vascular beds stimulated by vasoactive agents.24
The notion that the estrogen-elicited, CYP mediation of flow-induced dilation is a specific response that could be discerned in most instances only when NO synthesis is impaired or absent, is of significance, because it reveals the action of estrogen in recruitment of backup mechanisms under certain pathophysiological conditions. Indeed, it has been demonstrated that in physiological conditions, NO is still the primary mediator released to shear stress.2,3,33 In heart failure however, a condition characterized by reduced endothelial NO production, estrogen increased coronary blood flow and improved vascular dysfunction by opening of smooth muscle KCa channels via, most likely, an EDHF-mediated response.23
Mechanisms Responsible for the CYP-Mediated Responses by Incubation With 17ß-E2
Data shown in Figure 3 illustrate a framework of the 17ß-E2/ERs-PI3K/Akt-transcriptional mechanism of CYP mediation of flow-induced responses, as indicated by the fact that blockade of ERs (top panel) or PI3 kinase (middle panel) or inhibition of gene transcription (bottom panel) prevented the responses elicited by incubation of 17ß-E2. Estrogen is traditionally considered to have genomic and nongenomic effects via targeting its nuclear and membrane bound receptors, respectively. Although the rapid, nongenomic actions of estrogen are basically defined as membrane receptor dependent, an increasing body of evidence has demonstrated that gene transcription can also result from estrogen signaling initiated at the cell membrane.11,34 PI3K affects cellular functions via interaction with membrane-bound receptors.35 In this regard, cell membrane-bound ERs may be responsible for the recruitment of PI3K by estrogen, as indicated by the finding that immunostaining of cell membrane ER
is significantly increased in response to estradiol, concurrent with activation of PI3K.35 Such interaction between cell membrane ERs and activation of PI3K/Akt has been well characterized in connection with the nongenomic regulation of eNOS by estrogen.36 Our data provide evidence for a crucial role of ERs, that are most likely membrane-bound, and PI3K in the mediation of vascular responses.
Interestingly, our experiments indicated that acute administration of 17ß-E2 did not, but overnight incubation did, elicit CYP-mediated flow-induced dilation. The inhibitory effect of wortmannin and LY 294002 on CYP-mediated flow-induced dilation of arterioles was exerted only after incubation with the agents for 5 hours, and by 7 hours responses were completely abolished (data not shown). Collectively, these data suggest that it is a genomic effect of estrogen that potentiates CYP activity. This was further confirmed by the evidence that overnight incubation of the vessels with 17ß-E2 plus the transcriptional inhibitor DRB (Figure 3, bottom), prevented the CYP-mediated flow-induced dilation caused by incubation with 17ß-E2 alone. Moreover, a significant enhancement of EET production, indicative of a greater activity of the enzyme, in arterioles incubated for 8 hours with 17ß-E2 compared with those incubated without estrogen (Figure 5), further supports the conclusion that the responses are due to a transcriptionally based upregulation of CYP activity.
Molecular evidence of the role for PI3K/Akt pathway in the mediation of the responses is also provided by the fact that overnight incubation with 17ß-E2 significantly enhanced arteriolar phospho-Akt levels (Figure 4), a response that is consistent with the findings of others, which showed that overnight incubation with phytoestrogens significantly increases nuclear staining of phospho-Akt in cultured cardiac myocytes.7 Moreover, localization of phospho-Akt via immunohistochemistry indicates that endothelial cells are the main source of the estrogen-stimulated enhancement of phospho-Akt in these vessels (online Figure 4). This is of significance because not only is flow-induced dilation per se an endothelium-dependent response, but more importantly, the data provide histological evidence for a linkage between the estrogen-dependent increase in phospho-Akt and the shear stress-stimulated release of EETs, in addition to which prostaglandin-mediated flow-induced dilation in control conditions seems not to be affected significantly by inhibition of PI3K (online Figure 3). It is also of note that, although a period of at least 7 hours was necessary for the complete inhibition of CYP-mediated responses by wortmannin, as well as for the initiation of the response by estrogen, Akt phosphorylation in these vessels occurred after exposure to 17ß-E2 for only 30 minutes and lasted, at least, 8 hours (Figure 4). This seemingly paradoxical phenomenon reveals an integration of nongenomic and genomic regulation, which involves a rapid modulation of cellular kinase cascades, or second messengers, followed by gene transcription.37,38 Previous studies demonstrated that after estrogen binds to membrane receptors, followed by the activation of G proteins,34 multiple signaling pathways that have been linked to either the stimulation of gene transcription or posttranslational modification of proteins,39–41 are rapidly activated. A recent report provided evidence that in cultured endothelial cells, physiological concentrations of estradiol elicited substantial Akt phosphorylation within 5 minutes, followed by an upregulation of 250 genes after 40 minutes. This estrogen-induced increase in gene expression was dependent on PI3 kinase signaling, because LY294002 abolished the responses.14 The antiapoptotic effect of estrogen involving acute activation of PI3K/Akt and genomic regulation of endothelial function provides more evidence in favor of the dual action of estrogen.24 The findings are in agreement with our results showing that 17ß-E2 initiated a rapid phosphorylation of Akt in arterioles, followed by an enhancement of EET production in 8 hours, a response that was sensitive to inhibitors of estrogen receptors and PI3 kinase (Figures 4 and 5). Moreover, unlike ICI 182,780, which eliminated estrogen-elicited enhanced production of EETs, wortmannin, significantly but not completely, reversed the responses, implying that some other estrogen-dependent signaling pathway may also be involved. Thus, estrogen, through signaling, typically initiated at the membrane, activates the PI3K/Akt cascade. After this, the signaling pathways diverge, via nongenomic activation of downstream effectors, such as eNOS, and via phosphorylation of transcription factors to initiate genomic regulation. Based on the aforementioned studies, we interpret our findings to mean that binding of 17ß-E2, most likely to membrane receptors, rapidly activates the PI3K/Akt cascade, followed by a transcriptionally based regulation of CYP. These mechanisms we believe, form the basis of the phenomenon that activation of arteriolar phospho-Akt occurs already after 30 minutes exposure to 17ß-E2, but that 17ß-E2, as well as wortmannin, take several hours (overnight) to elicit, or reverse, respectively, CYP-mediated responses, a time necessary for target enzyme synthesis or degradation.
In conclusion, overnight incubation with physiological concentrations of estradiol elicits enhanced CYP-mediated flow-induced dilation, associated with an enhanced production of EETs in skeletal muscle arterioles of NO-deficient male and OV rats, via an ER-dependent, PI3K/Akt-mediated, transcriptional upregulation of CYP activity. These results also provide evidence of estrogen’s effects on gene transcription and vascular function that emanate from rapid and specific kinase signaling and the integration of cell membrane and nuclear effects of the hormone. Taken together, our results highlight the presence of a mechanism, by which estrogen could, especially in the absence or at a time of an impairment of endothelial NO synthesis, have beneficial effects by preventing or delaying the development of cardiovascular diseases, via compensatory upregulation of CYP/EDHF-mediated vascular dilator pathways.
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Acknowledgments |
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This study is supported by NIH HL 070653 (A. Huang), HL 68813 (D. Sun), and HL 43023 (G. Kaley). We gratefully acknowledge the generous supply of ICI 182,780 from Zeneca Pharmaceuticals.
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Footnotes |
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Original received July 1, 2003; resubmission received November 19, 2003; accepted November 21, 2003.
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References |
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