| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390
Correspondence: Address all correspondence and requests for reprints to: Philip W. Shaul, M.D., Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas Texas 75390. E-mail: pshaul{at}mednet.swmed.edu
 |
Abstract |
|---|
 Top Abstract I. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
localized to caveolae in endothelial cells, where they are coupled to eNOS in a functional signaling module. These observations, which emphasize dependence on cell surface-associated receptors, provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae. Estrogen binding to ER on the SRFC in caveolae leads to Gi activation, which mediates downstream events. The downstream signaling includes activation of tyrosine kinase-MAPK and Akt/protein kinase B signaling, stimulation of heat shock protein 90 binding to eNOS, and perturbation of the local calcium environment, leading to eNOS phosphorylation and calmodulin-mediated eNOS stimulation. These unique genomic and nongenomic processes are critical to the vasoprotective and atheroprotective characteristics of estrogen. In addition, they serve as excellent paradigms for further elucidation of novel mechanisms of steroid hormone action. I. Introduction
II. Estrogen, Nitric Oxide, and Vascular Function and Disease in Humans
A. Estrogen and human vascular disease
B. Sources of estrogens in humans
C. Long-term effects of estrogen on eNOS function
D. Short-term effects of estrogen on eNOS function
E. Role of estrogen receptors
III. Estrogen, NO, and Vascular Function and Disease in Animal Models
A. Estrogen, NO, and animal models of vascular disease
B. Role of ER in animal models of vascular disease
C. Long-term effects of estrogen on eNOS in animal models
D. Short-term effects of estrogen on eNOS in animal models
IV. Genomic Mechanisms of eNOS Regulation by Estrogen
A. Genomic regulation of eNOS expression by estrogen
B. Basis for eNOS up-regulation by estrogen
C. Other genomic mechanisms modifying eNOS function
V. Nongenomic Regulation of eNOS by Estrogen
A. Estrogen and eNOS activation
B. Localization of eNOS activation by estrogen to plasma membrane
C. Localization of ER-eNOS interaction to caveolae
D. Role of ERß in nongenomic eNOS activation
E. Coupling of plasma membrane ER to downstream signaling events
VI. Summary and Future Directions
|
I. Introduction |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
and ERß, will be described. Finally, the cellular and molecular mechanisms underlying the genomic and nongenomic regulation of eNOS by estrogen will be considered in depth. Further knowledge about the complex impact of estrogen on endothelial cell biology is needed to understand the normal actions of the hormone in the cardiovascular system, and to guide future considerations of hormone therapy to prevent or treat cardiovascular disease (4). |
II. Estrogen, Nitric Oxide, and Vascular Function and Disease in Humans |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
B. Sources of estrogens in humans
The estrogen compounds to which the vascular endothelium may be exposed in women are multiple, and they arise from both endogenous and exogenous sources. Before menopause, the major endogenously derived circulating estrogen compound is 17ß-estradiol (E2) produced by the ovary. Other endogenous estrogens and estrogen metabolites have also been described. After menses, when circulating E2 levels increase and begin to cycle, levels range from less than 0.36 nM during the follicular phase to 2.8 nM during midcycle. During pregnancy, estrogen levels rise to 70 nM due to placental production, which increases near term and further with the onset of parturition. After menopause, E2 concentrations fall to levels that are equivalent to those in males (0.04–0.21 nM). During menopause, the primary form of endogenous circulating estrogen is estrone derived peripherally from androstenedione by conversion in adipose tissue (15). Endothelial cells in both women and men may also be exposed to estrogens derived from the local conversion of testosterone or 
4-testosterone to E2 by aromatase (16). Aromatase, E2-hydroxysteroid dehydrogenase, and 17-ketoreductase enzyme activities were first demonstrated in rat arterial vascular smooth muscle (VSM) cells in cell culture (17), and more recently aromatase expression was noted in human VSM by in situ hybridization (18). Only trace levels were detected in samples from infants, and greater aromatase mRNA and activity were found in adult samples. In addition, studies in cultured human VSM demonstrated up-regulation of aromatase mRNA in response to cAMP, phorbol ester, or dexamethasone (18). Thus, there is evidence that aromatase expression in VSM is developmentally regulated and also modulated by multiple signal transduction pathways and by glucocorticoids. However, the relative impacts of circulating vs. locally derived estrogens on endothelial cell function have not yet been clearly elucidated.
In addition to endogenously derived estrogens, there are often important exogenous sources in humans. Contraception is frequently accomplished using a combination of ethinyl E2 and levonorgostrel or norethindrone. HRT is also often provided to postmenopausal women. This is frequently in the form of conjugated equine estrogens, and other oral and transdermal forms are also often considered. HRT frequently entails conjugated equine estrogens in combination with medroxyprogesterone acetate. In addition, selective estrogen receptor modulators, or SERMs, such as raloxifene are used for the treatment of osteoporosis, and it is likely that vascular-specific SERMS will also soon be available. Furthermore, phytoestrogens are a class of compounds found in certain plant-derived beverages and foods, and they have both estrogenic and antiestrogenic effects (14, 19, 20). As such, the endothelium may be exposed to a variety of forms and levels of estrogen compounds depending on the sex and age of the individual, his/her diet, and therapies that he/she may be receiving.
C. Long-term effects of estrogen on eNOS function
The onset of menopause provides a natural model of estrogen deprivation in which the effects of endogenous levels of the hormone on vascular function can be revealed. In studies of changes in brachial artery diameter after reactive hyperemia, which provides an assessment of flow-mediated, endothelium-dependent, and NO-dependent vasodilation, responses were greater in premenpausal vs. postmenopausal women (21). Similarly, determinations of forearm blood flow constrictor responses to NOS antagonism with L-NG-monomethyl arginine (L-NMMA), which also evaluate vascular NO activity, revealed greater constriction in premenopausal vs. postmenopausal women. Importantly, blood flow responses to the NO donor glyceryl trinitrate (GTN) were similar in the two study groups, indicating comparable VSM responses to NO. As might be predicted, the responses in postmenopausal women were comparable to those observed in men (Fig. 1
and Ref. 22).
|
and Ref. 25). In agreement with these findings, sex hormone deprivation after ovariectomy for uterine leiomyoma is associated with a decline in endothelium-dependent vasodilation, whereas the response to the NO donor sodium nitroprusside is unaltered (26). Although the exact sources of NO are undetermined, the enhanced endothelium-dependent vasodilation observed during periods of greater estrogen abundance during the menstrual cycle correlates with higher serum levels of the NO metabolites nitrate and nitrite and greater exhaled NO (23, 27, 28). Thus, although many other parameters may vary in parallel with the changes in estrogen status that accompany menopause or surgical ovariectomy or the menstrual cycle, there is a strong correlation between endogenous estrogen levels and the capacity for endothelial NO production in women.
|
). In addition, the interventions yield comparable increases in plasma NO levels (33). Interestingly, the inclusion of progestin in postmenopausal HRT appears to blunt the effects of estrogen on endothelial NO production (21), perhaps paralleling the findings noted above for variations in endothelial function during the menstrual cycle (24). Premenopausal women receiving estrogen replacement after ovariectomy also have enhanced endothelial function after therapy, and similar effects have been documented in young women receiving oral contraceptives (34). Long-term estrogen administration also improves vascular function in males, and this is at least partially mediated through endothelium-derived NO (35, 36). As such, prolonged exposure to exogenously derived estrogen has positive effects on endothelium-derived NO availability, which parallel those noted with altered endogenous levels and provides further evidence of potential long-term modulation of eNOS function by the hormone in humans.
|
). In addition, ethinyl E2 caused attenuation of vasoconstrictor responses to acetylcholine (Ach) when given 15 min before Ach (37). Experiments with intracoronary E2 administered in a manner yielding premenopausal levels did not demonstrate changes in basal coronary vasomotor tone, but there was greater vasodilatory response to Ach when E2 and Ach were given simultaneously. In these studies, E2 had no effect on vasodilation with sodium nitroprusside, suggesting that the hormone rapidly modifies the availability of endothelium-derived NO (38). Findings were similar when E2 effects on forearm vascular responses were assessed in postmenopausal women, with the exception that endothelium-independent vasodilation was also enhanced (39). In further studies in the coronary circulation of older women, the capacity of E2 to cause augmented Ach-induced responses was prevented by NOS antagonism, providing additional evidence that the underlying mechanism involves enhanced bioavailability of NO (40). In contrast to the observations made in postmenopausal women, intracoronary E2 administration to similarly aged men caused neither a change in basal vasomotor tone nor greater vasodilation with Ach tested 20 min later (41). However, the provision of iv conjugated estrogens 15 min earlier caused greater Ach-induced coronary blood flow and less cold-induced coronary vasoconstriction in men (42, 43). In a similar manner, phytoestrogens cause direct, rapid, NO-dependent dilation of the forearm vasculature of both men and women, which is comparable in degree to that observed with E2 (Fig. 5). Lower concentrations of phytoestrogen also potentiate the vasodilatory response to Ach. It is important to note that the threshold concentration of phytoestrogen yielding these effects does not greatly exceed those found in East Asian subjects in whom dietary phytoestrogen intake has been suggested to contribute to an extremely low incidence of atherosclerosis and coronary artery disease (44). These cumulative observations indicate that, in addition to the long-term effects of estrogen and estrogen-like compounds on eNOS action, there are short-term effects on the bioavailability of NO. Importantly, these effects may generally be comparable in men and women, and the estrogen formulation is a critical variable.
|
|
gene was found to have premature coronary artery disease at age 31 yr (45, 46). Flow-mediated vasodilation of the patient’s brachial artery was also attenuated, whereas vasodilation with sublingual nitroglycerin was intact, suggesting a diminished capacity for endothelial NO production. Interestingly, rapid vasodilation in response to sublingual E2 was normal (46). These observations may be explained by recent studies demonstrating that either ER or ERß can mediate rapid eNOS activation in cell culture. These findings are presented below in Section V.D. More direct studies of ER involvement in endothelial NO production have been performed in isolated human mammary artery segments. In precontracted rings obtained from men at the time of coronary artery bypass surgery, E2 at concentrations of 100 nM or greater caused relaxation that was prevented by either endothelium removal or NOS antagonism. The response to E2 was also attenuated 71% by the ER antagonist tamoxifen. Although issues relating to the concentration of E2 required for this in vitro response may be raised, these findings suggest that the short-term effects of E2 on endothelial NO bioavailability in humans are mediated by endothelial ER (47).
|
III. Estrogen, NO, and Vascular Function and Disease in Animal Models |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
Animal models have also been employed to demonstrate that estrogen has an important impact on the function of atherosclerotic arteries. In pioneering studies by Williams and colleagues (55, 56), both long-term (26 months) and brief (20 min) E2 treatment caused enhanced Ach-mediated vasodilation in atherosclerotic coronary arteries of female cynomologus monkeys. Similarly, long-term E2 administration in hypercholesterolemic swine led to the preservation of endothelium-dependent relaxation (57). Perhaps more importantly, there is considerable animal evidence that estrogen modifies the development of atherosclerosis. This includes studies in cholesterol-fed rabbits in which both ethinyl E2 and conjugated equine estrogen reduced atherosclerosis by 35% in the aortic arch and by 75–80% in the thoracic and abdominal aorta (58). Similarly, E2 administration to apolipoprotein E (apoE)-deficient mice decreased the atherosclerotic lesion area in both males and ovariectomized females. Importantly, neither the less biologically active 17-estradiol nor tamoxifen affected lesion development in ovariectomized females in the apoE deficiency paradigm, and the beneficial effects of 17ß-estradiol were only partially explained by changes in plasma lipoprotein levels (59). The latter variable has been elegantly controlled in studies of cholesterol-clamped rabbits in which the antiatherogenic properties of estrogen remain apparent. In addition, in the cholesterol-clamped rabbits the capacity of estrogen to prevent cholesterol accumulation in the vascular wall was abolished by balloon catheter injury, suggesting that an intact endothelium is required (Fig. 6) (60, 61). Furthermore, in the same model NOS inhibition reduced the atheroprotection afforded by estrogen, and comparable observations were made with the SERM levormeloxifene (Fig. 6). Moreover, the antiatherogenic effect of the hormone was preceded by an increase in NO production that was followed by diminished mononuclear-endothelial cell binding. It was noted additionally that the effect of E2 was to increase the basal release of NO, whereas NO release by Ach and NOS abundance was unaltered (62, 63). These cumulative observations suggest that the antiatherosclerotic characteristics of estrogen involve direct effects on the vascular endothelium, and that these effects may be primarily mediated by enhanced NO production unrelated to genomic effects of the hormone on eNOS expression.
|
-deficient and ERß-deficient mice (64, 65). Such observations suggested that either receptor isoform may be sufficient to mediate the effects of estrogen or that the effects are through a non-receptor-mediated process or through another unidentified ER. The first of these possibilities was addressed in recent studies of ER,ß double knockout mice. In the double knockouts, E2 did not prevent the injury-induced increase in vascular medial area. However, VSM cell proliferation continued to be attenuated by the hormone (66). In contrast, in experiments in rats employing nonselective pharmacological ER antagonism, it has been observed that the vasoprotective effects of E2 are completely inhibited (67). The role of ER in the antiatherogenic features of estrogen has also been addressed in a recent report. In mice deficient in apoE alone, E2 reduced the size of atherosclerotic lesions and also their histological complexity, as previously observed (59). Plasma cholesterol levels were decreased by E2, but the degree of the effects of the hormone on atherogenesis could not be explained solely on the basis of changes in the plasma lipid profile. In contrast, in mice with disrupted apoE and disrupted ER, E2 had a more modest effect on lesion size and total plasma cholesterol was unaltered. However, the complexity of the plaques was diminished by E2 despite the absence of ER (68). As such, certain mechanisms whereby estrogen alters the vascular response to injury and the progression of atherosclerosis are evidently mediated by ER or ERß. However, the bases for multiple other processes are yet to be determined. Furthermore, in contrast to the evidence that strongly implicates an important role for eNOS in the hormonal modulation of atherogenesis, studies are yet to be performed to clarify the specific contribution of eNOS to E2-induced changes in vascular injury responses.
C. Long-term effects of estrogen on eNOS in animal models
There is considerable evidence from investigations in intact animal models that long-term estrogen exposure enhances the capacity for endothelial NO production. For example, E2 treatment of rabbits for 4 d caused an increase in endothelium-dependent relaxation in femoral artery rings (69). Using NOS antagonism, it has also been shown that bioassayable endothelium-derived NO is higher in thoracic aortas isolated from female vs. male rats (70). The same group of investigators employed ER knockout mice to demonstrate that there is a significant association between the abundance of ER and the basal release of NO (71). In 1994 it was reported that steady-state eNOS mRNA levels are increased in guinea pig skeletal muscle during pregnancy and after E2 treatment. This may have been related to the induction of eNOS in endothelium, but it is notable that the cell specificity of eNOS up-regulation was not determined, and eNOS is expressed in skeletal myocytes (72, 73). The long-term effects of estrogen on eNOS expression have been demonstrated in studies of the uterine circulation in sheep. It was reported in 1996 that after 3 d of systemic E2 treatment, there was enhanced NO-dependent, endothelium-dependent relaxation, which was related to greater NOS enzymatic activity (74). It has since been shown that eNOS abundance is up-regulated in uterine artery endothelium after both prolonged (days) and brief (2 h) E2 treatment (75, 76). Although E2-related effects on eNOS do not occur in certain systemic arteries in the sheep (75), studies in rats have demonstrated dramatic eNOS up-regulation in cerebral microvessels after chronic E2 treatment. Interestingly, the eNOS responses to E2 in the cerebral microvessels were similar in female and male rats (77). In addition, studies with knockout mice have demonstrated that the E2-induced up-regulation in eNOS expression in the cerebral microvessels is mediated by ER, and that parallel changes occur in the abundance of cyclooxygenase type 1, which is the rate-limiting enzyme in prostacyclin production (Fig. 7) (78). Furthermore, work in porcine coronary arteries has shown that the effects of E2 are direct and not mediated by changes in blood flow, since isolated ring segments displayed greater NO-dependent relaxation after 18–22 h exposure to E2 in vitro (79).
|
-zearalenol causes enhanced endothelial NO production, and the effect is fully prevented by the ER antagonist ICI 182,780, indicating that it is mediated by ER binding (82). Chronic exposure to phytoestrogen also improves endothelial dysfunction induced by ovariectomy in rats (83).
D. Short-term effects of estrogen on eNOS in animal models
Experiments in isolated segments of rabbit coronary artery, rat superior mesenteric artery, and rat aorta from female animals have demonstrated that physiological levels of E2 cause rapid, NO-dependent, endothelium-dependent relaxation or rapid, NO-dependent inhibition of platelet aggregation (84, 85, 86). However, a considerable number of additional works have alternatively demonstrated NO-independent, endothelium-independent E2-mediated relaxation (87, 88, 89). The varying results related to endothelial NO production may be due to differences in the species or vascular beds under study, as well as varying usage of precontraction. In addition, the estrogen status of the animal is critically important. Collins and colleagues (84) demonstrated that coronary artery rings from oophorectomized, estrogen-treated, and acutely estrogen-withdrawn rabbits display robust endothelium-dependent, NO-dependent relaxation to E2, whereas rings from oophorectomized, untreated or oophorectomized, estrogen-maintained rabbits display more modest responses (Fig. 8). Interestingly, the SERM raloxifene caused rapid NO-dependent, endothelium-dependent relaxation of coronary artery rings from both male and female rabbits studied under conditions that yielded only NO-independent responses to E2 (88). In addition, 30-min in vitro exposure to phytoestrogen restored NO-mediated relaxation in pulmonary arteries isolated from chronically hypoxic rats (90). Thus, paralleling the findings related to endothelial NO-mediated responses in humans, studies in animals indicate that estrogen causes both long-term up-regulation of eNOS expression and rapid enhancement of eNOS enzyme activation.
|
|
IV. Genomic Mechanisms of eNOS Regulation by Estrogen |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
expression in early-passage HUVEC and diminished levels of expression with additional passage (92). Further studies in ovine endothelial cells demonstrated that E2 induces increases in NOS enzymatic activity and eNOS protein abundance at concentrations of 0.1 nM E2 or greater, and that these changes are accompanied by increases in eNOS mRNA levels. Transient transfection assays with a specific estrogen-responsive reporter system also demonstrated that endothelial ERs are capable of estrogen-induced transcriptional transactivation (93). Similar observations have also been made in human osteoblast-like cells, consistent with other data suggesting that NO may mediate estrogen action in bone (94). eNOS expression in neonatal and adult cardiac myocytes is also up-regulated by E2 in an ER-dependent manner, potentially explaining some of the cardioprotective effects of the hormone (95). In addition, estrogen treatment caused an increase in eNOS mRNA abundance in LNCaP human prostate carcinoma cells within 2–4 h of exposure. Interestingly, estrone sulfate, the most abundant circulating estrogen that may serve as a prehormone for the terminal biologically active estrogen E2 in men, also up-regulated eNOS mRNA in LNCap cells, and the response required 48 h, suggesting conversion to E2 (96).
B. Basis for eNOS up-regulation by estrogen
Work in human endothelial EA.hy 926 cells indicates that E2 or the more stable 17-ethinyl E2 enhances eNOS protein and mRNA abundance in the absence of changes in the stability of eNOS mRNA. Nuclear run-on assays showed that the increase in eNOS mRNA abundance is related to greater eNOS gene transcription. The impact of 17-ethinyl E2 on eNOS gene transcription was further evaluated in transient transfection studies using a 1.6-kb human eNOS promoter fragment, which lacks classical estrogen response elements (EREs). Promoter activity was enhanced to a degree comparable to the increase in eNOS mRNA levels, and EMSAs suggested greater DNA-protein complex formation involving a putative Sp1 binding element after estrogen treatment. However, immunodepletion or supershift analyses were not performed to specifically identify the nuclear protein that is involved (97). The role of ER subtypes in eNOS gene activation by E2 has been elucidated in neonatal rat cardiac myocytes. eNOS up-regulation by E2 was fully prevented by the ERß-specific antagonist RR-tetrahydrochrysene (THC), indicating a primary role for that subtype (Fig. 9) (98). Further work in cultured endothelial cells indicated that eNOS down-regulation by TNF, which is due to enhanced eNOS mRNA degradation, is prevented by E2, and that this mechanism is also ER dependent (99). Thus, there is transcriptional regulation of eNOS expression by E2 through mechanisms that do not involve classical ERE-mediated processes, and additional nontranscriptional mechanisms may be important under certain conditions.
|
In addition to the relative level of eNOS enzymatic activity, the ultimate bioavailability of NO as a signaling molecule is modified by the degree of scavenging by reactive oxygen species such as superoxide. In human endothelial cells, superoxide levels are dictated by the production of the radical by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and its degradation by superoxide dismutase (SOD). In recent studies in HUVEC, E2 caused a 60% decrease in the expression of the protein and mRNA for the NADPH oxidase subunit gp91phox. There was also a less dramatic, but significant, E2-induced decline in mRNA abundance for p22phox, which, together with gp91phox, constitute the membrane-bound cytochrome b558 portion of the NADPH oxidase complex. The two cytosolic activating factors of the complex, p47phox and p67phox, were also down-regulated by E2, and the net effect was an approximately 50% diminution in the capacity to produce superoxide. In addition, the effects of E2 on NADPH oxidase subunit expression and superoxide production were prevented by ER antagonism. Heat shock protein 90 (HSP90) and eNOS expression were also up-regulated by E2. In contrast, levels of expression of the reactive oxygen species-metabolizing enzymes glutathione-peroxidase, copper/zinc SOD, manganese SOD, and catalase were not affected by E2 (Fig. 10 and Ref. 104). Thus, in addition to up-regulating eNOS abundance, E2 inhibits the expression of NADPH oxidase in endothelial cells, thereby potentially increasing the bioavailability of NO through more than one process.
|
|
V. Nongenomic Regulation of eNOS by Estrogen |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
is expressed in cultured endothelial cells (Fig. 11 and Refs. 105, 106, 107). It has also been shown that the overexpression of ER in endothelial cells causes enhancement of the acute response to E2 that is blocked by ER antagonism and specific to E2 vs. other agonists (Fig. 12). In addition, the enhanced response is dependent on the ER hormone binding domain. In COS-7 cells, which do not constitutively express ER or eNOS, the acute stimulation of eNOS by E2 can be demonstrated after the cotransfection of ER and eNOS cDNAs (107). Raloxifene also activates endothelial cell eNOS within 10 min, and the process is ER dependent since it is abolished by ICI 182,780 (108). eNOS activation by E2 has also been observed in human bronchiolar epithelial cells, which also constitutively express the enzyme and ER, suggesting that mechanisms similar to those delineated in the vasculature may play a role in hormonal modulation of airway function (109).
|
|
). These data indicate that the acute stimulation of eNOS by E2 and ER entails the activation of tyrosine kinase/MAPK (107). There is also evidence of a role for the recruitment of the PI3-kinase-protein kinase B/Akt pathway. In studies of human endothelial cells, E2-induced eNOS activation was prevented by the PI3-kinase inhibitor, LY294002, and E2 caused rapid Akt phosphorylation on serine 473. E2 also caused eNOS phosphorylation on serine 1177, which is a critical residue for eNOS activation and modification of the sensitivity to cellular calcium levels (Fig. 14). In addition, expression of a kinase-deficient, dominant-negative Akt prevented E2-stimulated NO production (110, 111, 112). Additional work suggests that E2-induced PI3-kinase/Akt signaling is mediated exclusively by ER and not ERß, and that it entails direct interaction between ER and the p85 subunit of PI3-kinase (111, 112). Recent examinations of the signal transduction events by which raloxifene stimulates eNOS have also implicated involvement of MAPK and PI3-kinase/Akt signaling that is mediated by ER (113).
|
|
Additional mechanisms of eNOS regulation in addition to those involving protein kinase-mediated phosphorylation events and changes in calcium homeostasis have also been investigated. In studies of HUVEC, inhibitors of HSP90 function prevented E2-stimulated NO release and cGMP production. E2 also was found to induce HSP90-eNOS association, and this event was prevented by ER antagonism, providing further evidence for a role for HSP90 in eNOS activation by E2 (116). Thus, the short-term effects of estrogen on eNOS that are central to cardiovascular physiology are mediated by ER functioning in a novel, nongenomic manner, and multiple signal transduction events are likely to be involved.
B. Localization of eNOS activation by estrogen to plasma membrane
Studies employing immunoidentification or conjugated estrogen have indicated that a subpopulation of ER may be associated with the cell surface in certain cell types (117, 118). In fact, immunofluorescence experiments suggest that the ligand binding domain of the ER may actually reside on the extracellular face of the plasma membrane (119). As mentioned above, there is strong evidence that eNOS is targeted to the endothelial plasma membrane, particularly to caveolae, which are specialized, cholesterol-rich lipid-ordered domains that compartmentalize signal transduction. eNOS trafficking to caveolae is dependent upon the myristoylation and palmitoylation of the protein (100, 120, 121, 122). Initial determinations of the subcellular site of interaction between ER and eNOS involved studies of plasma membranes isolated from ovine endothelial cells, in which eNOS stimulation was evaluated by measuring 3H-L-arginine conversion to 3H-L-citrulline (123). In the absence of added calcium, calmodulin, or cofactors, 10-8M E2 (15 min) caused a 92% increase in NOS activity compared with basal levels, whereas 17-estradiol had no effect. Maximal NOS activity was assessed by replacing E2 with a mixture of calcium, calmodulin, and cofactors, yielding a 170% rise in activity compared with no additives; E2 (10-8M) did not enhance this activity (Fig. 15A). These observations indicated that all of the signal transduction machinery necessary for eNOS stimulation by E2 is associated with the plasma membrane. Since E2 alone activated the enzyme to approximately half of maximal levels, the response is quite robust. Time course experiments with E2 alone further revealed a progressive, linear increase in NOS activity during the first 30 min of incubation, followed by a plateau (Fig. 15B). This contrasted with NOS activity with added calcium, calmodulin, and cofactors, which displayed linearity with time for at least 120 min (123). These data suggest that the availability of one or more of these molecules is limited in the isolated membranes; in contrast, in intact cells there are most likely mechanisms that replenish these factors in the locale of the plasma membrane.
|
). In addition, antibody to the ligand binding domain of ER (TE111) blocked E2-stimulated NOS activation, whereas unrelated IgG had no effect (Fig. 15D). These observations indicated that the response to E2 is mediated by an ER or ER-like protein associated with the endothelial cell plasma membrane. Immunoidentification experiments, which compared the plasma membrane ER with cytosolic and nuclear ER using antibodies directed against three different ER epitopes, were then performed. Antibodies directed against amino acids 495–595 (AER320), 302–553 (TE111), or 120–170 of human ER (AER304) all detected a single 67-kDa protein species in endothelial cell plasma membranes that was identical in size to the protein detected in nuclear and cytosolic fractions (Fig. 16A). Confirmatory studies were done involving the localization of epitope-tagged ER (ER-myc) transiently transfected into COS-7 cells. Whereas antibody to ER revealed no signal in sham-transfected cells, there was positive signal for ER of comparable size in nucleus, cytosol, and plasma membrane from cells transfected with ER-myc. In parallel, immunoblot analysis with antibody to the myc tag revealed no signal in sham-transfected cells, but a protein of similar size was detected in the nucleus, cytosol, and plasma membranes of cells expressing the tagged receptor (Fig. 16B). These cumulative findings indicate that E2-stimulated eNOS activity is mediated by a subpopulation of ER that is associated with the endothelial plasma membrane (123). Independent investigations demonstrating eNOS activation by membrane-impermeant E2 conjugated to BSA have provided additional evidence of eNOS signaling by cell surface ER (110, 115).
|
-stimulated eNOS activity was evaluated in a reconstitution paradigm in COS-7 cells. Plasma membranes from cells expressing eNOS and ER displayed rapid ER-mediated NOS stimulation, whereas membranes from cells expressing eNOS alone or ER plus myristoylation-deficient mutant eNOS were insensitive. In fact, membranes from cells expressing myristoylation-deficient mutant eNOS and ER displayed a decline in NOS activity with E2 that was partially reversed by ICI 182,780. A myristoylation-deficient mutant eNOS is minimally directed to the plasma membrane but unaltered in enzymatic activity (120), these findings indicate that both normal plasma membrane targeting of eNOS and localization of ER to that site are required for eNOS activation by E2 (123).
C. Localization of ER-eNOS interaction to caveolae
Since plasma membrane eNOS is exclusively localized to caveolae (120), further experiments were done to determine whether ER protein is also associated with this subfraction of endothelial cell plasma membranes. ER protein was detected in caveolae, and it was also detected, but to a lesser extent, in the noncaveolae fraction of the plasma membrane (Fig. 17A). Experiments were then performed to evaluate the capacity of E2 to activate eNOS in isolated caveolae and noncaveolae fractions. In the absence of added calcium, calmodulin, or cofactors, there was no measurable NOS activity in the noncaveolae fraction under basal conditions or with E2 added. Basal NOS activity was also below detection limits in caveolae membranes. However, 10-8 M E2 caused robust activation of NOS in caveolae membranes, and this effect was prevented by ICI 182,780 (Fig. 17B). These data strongly indicate that ER and all of the additional molecular machinery necessary for E2-mediated activation of eNOS exist in a functional signaling module in endothelial caveolae. Since ER was found in both caveolae and noncaveolae fractions and eNOS is solely in caveolae (120), the specificity of ER coupling to eNOS to caveolae is evidently due to the localization of the effector, and not the receptor, in the microdomain. Furthermore, the effect of E2 on eNOS in caveolae is prevented by calcium chelation (123), suggesting that ER activation in caveolae modifies the local calcium environment.
|
and ERß expression have both been observed in endothelial cells in multiple paradigms (14). To determine whether endogenous ERß plays a role in nongenomic eNOS activation, studies have been performed recently in ovine endothelial cells that display constitutive expression of both isoforms. It was found that a subpopulation of endogenous ERß is localized to the endothelial cell plasma membrane, overexpression of ERß in endothelial cells enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERß-selective antagonist THC. In addition, eNOS activation through ERß was reconstituted and shown to occur independent of ER in COS-7 cells (123A ).
Further experiments were done in isolated cell membranes to delineate the localization of ERß-eNOS coupling. It was observed that THC blunts E2 activation of eNOS in isolated endothelial cell plasma membranes. In addition, ERß protein was detected, THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERß-eNOS coupling was recapitulated in caveolae from transfected cells (123A ). Thus, both ER, as described above, and ERß have nongenomic action in endothelial cell caveolae to regulate eNOS activity in this specialized subcellular domain.
E. Coupling of plasma membrane ER to downstream signaling events
The basis by which signaling events are initiated by plasma membrane ER is perhaps best understood in the context of the known processes underlying eNOS activation by classical agonists such as Ach and bradykinin. These agents activate specific plasma membrane-associated G protein-coupled receptors (GPCRs) (124, 125). G proteins are heterotrimers of -, ß-, and 
-subunits (Gß) that dissociate into G and Gß upon GPCR stimulation, after which activated G and/or Gß modulate the activity of downstream effector molecules. Based on sequence and functional similarities, the -subunits are divided into four subfamilies: Gs, Gi, Gq, and G12/13 (126, 127). Within this context, the potential role of G proteins in the coupling of ER to downstream signaling events has been investigated in endothelial cells (128). The first approach employed was to measure E2-stimulated eNOS activity in the absence or presence of pertussis toxin treatment, which inhibits G function by causing ADP ribosylation of a conserved cysteine at the fourth position (129). Intact endothelial cells were pretreated with vehicle or pertussis toxin and exposed to either the known GPCR agonist Ach (10-5 M) or E2 (10-8 M) for 15 min. In the absence of pertussis toxin, Ach and E2 caused comparable eNOS stimulation. As expected, eNOS activation by Ach was fully blocked by pertussis toxin. Similarly, eNOS stimulation by E2 was prevented by pertussis toxin (Fig. 18A). In contrast, pertussis toxin did not prevent eNOS activation by the calcium ionophore A23187. The inhibition of ER-mediated eNOS activation by pertussis toxin implicates Gi subfamily members only, of which Gi is expressed in endothelial cells and Go is not (127, 129, 130).
|
). E2-stimulated eNOS activity was not affected by GTP or GTPS (128).
To confirm the observations made in endothelial cells and to provide a model system amenable to manipulation by cotransfection, the effect of pertussis toxin on E2-stimulated NOS activity was determined in COS-7 cells transfected with eNOS and ER (Fig. 18C). As observed in primary endothelial cells, E2 treatment (10-8 M for 15 min) caused eNOS stimulation that was completely blocked by pertussis toxin; in contrast, cells transfected with eNOS alone were not responsive to E2 (107). These cumulative data indicate that Gi mediates estrogen stimulation of eNOS. Pertussis toxin also prevented E2-mediated phosphorylation of MAPK in endothelial cells, indicating that G protein coupling occurs proximal to tyrosine kinase-MAPK activation in the series of events leading to eNOS stimulation (128).
Potential interactions between plasma membrane ER and G proteins have been evaluated in coimmunoprecipitation studies using COS-7 cells transfected with ER and either Gi2, Gq, or Gs (Fig. 19A). Immunoprecipitation was performed with ER antibody on plasma membranes from cells treated with vehicle or 10-8 M E2 for 20 min. In plasma membranes from quiescent cells, Gi, Gq, and Gs were minimally coimmunoprecipitated with ER. However, the association of Gi with ER was markedly greater after E2 stimulation. In contrast, the association of Gq and Gs with ER remained negligible after E2 treatment. Identical findings were obtained in endothelial cells (Fig. 19B). Thus, in both an overexpression system and at constitutive levels of abundance, ER activation by agonist leads to interaction between the receptor and Gi. All known GPCRs have seven-transmembrane spanning domains linked by alternating intracellular and extracellular loops, and extensive experimentation indicates that the GPCR intracellular domains function in the direct signal propagation to Gß (126, 131). Since the structure of ER is entirely different from any known GPCR, it is unlikely that ER-Gi interaction is direct. Alternatively, ER may be coupled to a classical GPCR that interacts directly with Gi. There is evidence that cross-talk of this type can occur to enable non-GPCR to perform G protein-mediated functions, such as the direct protein-protein coupling of -aminobutyric acid A receptors to dopamine D5 receptors in hippocampal neurons. The cross-talk involves specific domains on both the non-GPCR undergoing ligand activation and the GPCR interacting with G protein (132). The third possibility is that ER-Gi interaction requires an intermediary protein that is not a GPCR (128).
|
- or Gß-subunits modulate the activity of downstream effectors (130). The potential roles of activated G or Gß in E2-induced activation of eNOS have been distinguished by assessing the impact of overexpression of Gi2 in COS-7 cells expressing ER and eNOS (Fig. 20A). Whereas basal eNOS activity was unchanged by cotransfection with Gi2, Gi2 overexpression augmented E2-mediated eNOS stimulation by 3-fold, and pertussis toxin completely blocked the enhanced response. Gi1 overexpression had similar effects. The role of activated endogenous Gi was further substantiated in experiments assessing the effect of overexpression of a protein regulator of G protein signaling (RGS), RGS4 (Fig. 20B). RGS4 attenuates signaling by accelerating the GTPase activity of members of the Gi and Gq subfamilies, but not that of Gs or G12/13 (133). In COS-7 cells expressing ER and eNOS that were cotransfected with RGS4 cDNA, E2-mediated eNOS stimulation was decreased by 90% compared with sham-transfected cells. These cumulative observations indicate that activated Gi mediates the downstream signaling processes that ultimately lead to eNOS activation. In addition to explaining the proximal events underlying nongenomic effects of estrogen in endothelial cells, these observations provide further support of a role for G proteins in nongenomic actions of estrogen in other cell types such as osteoblasts and macrophages (134, 135). There is also accumulating evidence that certain nongenomic actions of glucocorticoids, vitamin D3, and testosterone involve steroid hormone receptors acting through pertussis toxin-sensitive mechanisms (136, 137, 138, 139). It is proposed that the processes by which plasma membrane ER are coupled to Gi in endothelial cells constitute not only the basis of nongenomic estrogen action in vascular and nonvascular cells, but also a means by which other steroid hormones may have important nonnuclear effects.
|
|
VI. Summary and Future Directions |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
localized to caveolae in endothelial cells, where they are coupled to eNOS in a functional signaling module (Fig. 21). Emphasizing the dependence on cell surface-associated receptors, these observations provide evidence for the existence of a steroid receptor fast-action complex, or SRFC, in caveolae. Estrogen binding to ER within caveolae leads to Gi activation, which mediates downstream events. The downstream signaling includes activation of tyrosine kinase-MAPK and Akt/protein kinase B signaling, stimulation of HSP90 binding to eNOS, and perturbation of the local calcium environment, ultimately leading to eNOS phosphorylation and calmodulin-mediated eNOS stimulation. The resulting combination of genomic and nongenomic mechanisms by which estrogen modulates eNOS plays a critical role in vascular health.
|
may be extracellular (119), the orientation of plasma membrane ER is yet to be elucidated. The mechanisms by which ER is membrane associated and the processes regulating the relative number of cell surface ER are not well understood. The basis for ER-Gi interaction, which may involve a classical GPCR or an alternative intermediate protein, is also entirely unknown. Furthermore, the proximal signal transduction events following Gi activation deserve in-depth study. Our present knowledge of eNOS modulation by estrogen typifies but a fraction of what may ultimately be a total of four categories of ER action. These categories are 1) membrane-initiated, nongenomic actions; 2) membrane-initiated, genomic actions; 3) non-membrane-initiated, nongenomic actions; and 4) non-membrane-initiated, genomic actions. When one considers that there are not only ER-dependent but also ER-independent mechanisms of estrogen response in certain paradigms, there are actually eight categories of possible estrogen action. These categories should be kept foremost in mind as we probe further into the bases for estrogen effects on the vasculature and other nonreproductive target organs.
Importantly, within the eNOS realm and elsewhere, the physiological and pathophysiological impact of the different modes of estrogen and ER action is yet to be delineated in intact model systems. It is only through such focused efforts that deeper understanding will be gained about processes including novel means of genomic estrogen action and ER function in a SRFC in caveolae. It is further anticipated that we will discover that other steroid hormone responses are either fully compartmentalized or at least initiated in SRFC on the plasma membranes of other cell types.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: Ach, Acetylcholine; apoE, apolipoprotein E; E2, 17ß-estradiol; eNOS, endothelial isoform of NOS; ER, estrogen receptor; ERE, estrogen response element; GDPßS, guanosine 5'-O-(2-thiodiphosphate); GPCR, G protein-coupled receptor; GTN, glyceryl trinitrate; HRT, hormone replacement therapy; HSP90, heat shock protein 90; HUVEC, human umbilical vein endothelial cells; L-NAME, nitro-L-arginine methyl ester; L-NMMA, L-NG-monomethyl arginine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOS, NO synthase; PI3-kinase, phosphatidylinositol 3-kinase; RGS, regulator of G protein signaling; SERM, selective ER modulator; SOD, superoxide dismutase; SRFC, steroid receptor fast-action complex; THC, tetrahydrochrysene; VSM, vascular smooth muscle.
|
References |
|---|
|
TopAbstractI. IntroductionII. Estrogen, Nitric Oxide,...III. Estrogen, NO, and...IV. Genomic Mechanisms of...V. Nongenomic Regulation of...VI. Summary and Future...References |
|---|
-adrenergic responses in resistance vessels during the menstrual cycle in healthy women. J Clin Endocrinol Metab 86:2499–2504-dihydroequilin sulfate, a conjugated equine estrogen, and ethynylestradiol on atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol 15:837–846-deficient mice. Nat Med 3:545–548[CrossRef][Medline]
, ß (double) knockout mice. Circ Res 89:534–539 is a major mediator of 17ß-estradiol’s atheroprotective effects on lesion size in apoE-/- mice. J Clin Invest 107:333–340[Medline]
. J Appl Physiol 91:2391–2399-zearalenol reverses endothelial dysfunction induced by oophorectomy in rats. Lab Invest 81:125–132[Medline]
through estrogen receptor mediated system. Life Sci 69:1651–1660[CrossRef][Medline]
mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401–406[Medline]
and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319(i). J Biol Chem 276:27071–27076-aminobutyric acid A receptors. Nature 403:274–280[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  I. Romieu, A. Fabre, A. Fournier, F. Kauffmann, R. Varraso, S. Mesrine, B. Leynaert, and F. Clavel-Chapelon Postmenopausal hormone therapy and asthma onset in the E3N cohort Thorax, February 8, 2010; (2010) thx.2009.116079v1. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. H. Kang, J. L. Wiggs, B. A. Rosner, S. E. Hankinson, W. Abdrabou, B. J. Fan, J. Haines, and L. R. Pasquale Endothelial Nitric Oxide Synthase Gene Variants and Primary Open-Angle Glaucoma: Interactions with Sex and Postmenopausal Hormone Use Invest. Ophthalmol. Vis. Sci., February 1, 2010; 51(2): 971 - 979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Schreckenberg, S. Wenzel, R. M. da Costa Rebelo, A. Rothig, R. Meyer, and K.-D. Schluter Cell-Specific Effects of Nitric Oxide Deficiency on Parathyroid Hormone-Related Peptide (PTHrP) Responsiveness and PTH1 Receptor Expression in Cardiovascular Cells Endocrinology, August 1, 2009; 150(8): 3735 - 3741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. T. Hess, M. W. Foster, and J. S. Stamler Assays for S-Nitrosothiols and S-Nitrosylated Proteins and Mechanistic Insights Into Cardioprotection Circulation, July 21, 2009; 120(3): 190 - 193. [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ponnuswamy, E. Ostermeier, A. Schrottle, J. Chen, P. L. Huang, G. Ertl, B. Nieswandt, and P. J. Kuhlencordt Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase Am. J. Pathol., June 1, 2009; 174(6): 2400 - 2410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Balligand, O. Feron, and C. Dessy eNOS Activation by Physical Forces: From Short-Term Regulation of Contraction to Chronic Remodeling of Cardiovascular Tissues Physiol Rev, April 1, 2009; 89(2): 481 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Peano, J. S. Crabtree, B. S. Komm, R. C. Winneker, and H. A. Harris Effects of Various Selective Estrogen Receptor Modulators with or without Conjugated Estrogens on Mouse Mammary Gland Endocrinology, April 1, 2009; 150(4): 1897 - 1903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. McCurnin, R. A. Pierce, B. C. Willis, L. Y. Chang, B. A. Yoder, I. S. Yuhanna, P. L. Ballard, R. I. Clyman, N. Waleh, W. Maniscalco, et al. Postnatal Estradiol Up-regulates Lung Nitric Oxide Synthases and Improves Lung Function in Bronchopulmonary Dysplasia Am. J. Respir. Crit. Care Med., March 15, 2009; 179(6): 492 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Farsetti, A. Grasselli, S. Bacchetti, C. Gaetano, and M. C. Capogrossi The telomerase tale in vascular aging: regulation by estrogens and nitric oxide signaling J Appl Physiol, January 1, 2009; 106(1): 333 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shibasaki, D. A. Low, S. L. Davis, and C. G. Crandall Nitric oxide inhibits cutaneous vasoconstriction to exogenous norepinephrine J Appl Physiol, November 1, 2008; 105(5): 1504 - 1508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Woldesenbet, C R Rosenfeld, O Ramilo, S Johnson-Welch, and J M Perlman Severe neonatal hypoxic respiratory failure correlates with histological chorioamnionitis and raised concentrations of interleukin 6 (IL6), IL8 and C-reactive protein Arch. Dis. Child. Fetal Neonatal Ed., November 1, 2008; 93(6): F413 - F417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zeng and Q. Xu eNOS-ER{alpha} Complex Goes to Telomerase Circ. Res., July 3, 2008; 103(1): 10 - 12. [Full Text] [PDF]
|
|
|
|
|
|
A. Grasselli, S. Nanni, C. Colussi, A. Aiello, V. Benvenuti, G. Ragone, F. Moretti, A. Sacchi, S. Bacchetti, C. Gaetano, et al. Estrogen Receptor-{alpha} and Endothelial Nitric Oxide Synthase Nuclear Complex Regulates Transcription of Human Telomerase Circ. Res., July 3, 2008; 103(1): 34 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Zicari, C. Centonze, M. Realacci, B. Buchetti, A. Pietropolli, and C. Ticconi Estradiol 17-{beta} and Progesterone Modulate Inducible Nitric Oxide Synthase and High Mobility Group Box 1 Expression in Human Endometrium Reproductive Sciences, July 1, 2008; 15(6): 559 - 566. [Abstract] [PDF]
|
|
|
|
 |
|
 C. M. Klinge, N. S. Wickramasinghe, M. M. Ivanova, and S. M. Dougherty Resveratrol stimulates nitric oxide production by increasing estrogen receptor {alpha}-Src-caveolin-1 interaction and phosphorylation in human umbilical vein endothelial cells FASEB J, July 1, 2008; 22(7): 2185 - 2197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Buys, P. Sips, P. Vermeersch, M. J. Raher, E. Rogge, F. Ichinose, M. Dewerchin, K. D. Bloch, S. Janssens, and P. Brouckaert Gender-specific hypertension and responsiveness to nitric oxide in sGC{alpha}1 knockout mice Cardiovasc Res, July 1, 2008; 79(1): 179 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Raulic, Y. Ramos-Valdes, and G. E DiMattia Stanniocalcin 2 expression is regulated by hormone signalling and negatively affects breast cancer cell viability in vitro J. Endocrinol., June 1, 2008; 197(3): 517 - 529. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Z. Sheng, F. Arshad, J. E. Braun, and A. P. Braun Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1531 - C1541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Kan, J.-T. Hsu, Z.-F. Ba, M. G. Schwacha, J. Chen, M. A. Choudhry, K. I. Bland, and I. H. Chaudry p38 MAPK-dependent eNOS upregulation is critical for 17{beta}-estradiol-mediated cardioprotection following trauma-hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2627 - H2636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Lim and L. Kobzik Sexual Tension in the Airways: The Puzzling Duality of Estrogen in Asthma Am. J. Respir. Cell Mol. Biol., May 1, 2008; 38(5): 499 - 500. [Full Text] [PDF]
|
|
|
|
|
|
K. Chang and Lubo Zhang Review Article: Steroid Hormones and Uterine Vascular Adaptation to Pregnancy Reproductive Sciences, April 1, 2008; 15(4): 336 - 348. [Abstract] [PDF]
|
|
|
|
 |
|
 C. Pinna, A. Cignarella, P. Sanvito, V. Pelosi, and C. Bolego Prolonged Ovarian Hormone Deprivation Impairs the Protective Vascular Actions of Estrogen Receptor {alpha}Agonists Hypertension, April 1, 2008; 51(4): 1210 - 1217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Si and D. Liu Genistein, a Soy Phytoestrogen, Upregulates the Expression of Human Endothelial Nitric Oxide Synthase and Lowers Blood Pressure in Spontaneously Hypertensive Rats J. Nutr., February 1, 2008; 138(2): 297 - 304. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Straub The Complex Role of Estrogens in Inflammation Endocr. Rev., August 1, 2007; 28(5): 521 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Simoncini, X-D. Fu, A. Caruso, S. Garibaldi, C. Baldacci, M.S. Giretti, P. Mannella, M.I. Flamini, A.M. Sanchez, and A.R. Genazzani Drospirenone increases endothelial nitric oxide synthesis via a combined action on progesterone and mineralocorticoid receptors Hum. Reprod., August 1, 2007; 22(8): 2325 - 2334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. K. Sumanasekera, G. U. Sumanasekera, K. A. Mattingly, S. M. Dougherty, R. S. Keynton, and C. M. Klinge Estradiol and dihydrotestosterone regulate endothelial cell barrier function after hypergravity-induced alterations in MAPK activity Am J Physiol Cell Physiol, August 1, 2007; 293(2): C566 - C573. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Mann, D. J. Rowlands, F. Y.L. Li, P. de Winter, and R. C.M. Siow Activation of endothelial nitric oxide synthase by dietary isoflavones: Role of NO in Nrf2-mediated antioxidant gene expression Cardiovasc Res, July 15, 2007; 75(2): 261 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Kumar, Q. Wu, K. L. Chambliss, I. S. Yuhanna, S. M. Mumby, C. Mineo, G. G. Tall, and P. W. Shaul Direct Interactions with G{alpha}i and G{beta}{gamma} Mediate Nongenomic Signaling by Estrogen Receptor {alpha} Mol. Endocrinol., June 1, 2007; 21(6): 1370 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Haas, M. R. Meyer, U. Schurr, I. Bhattacharya, R. Minotti, H. H. Nguyen, A. Heigl, M. Lachat, M. Genoni, and M. Barton Differential Effects of 17{beta}-Estradiol on Function and Expression of Estrogen Receptor {alpha}, Estrogen Receptor {beta}, and GPR30 in Arteries and Veins of Patients With Atherosclerosis Hypertension, June 1, 2007; 49(6): 1358 - 1363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Yamaleyeva, P. E. Gallagher, S. Vinsant, and M. C. Chappell Discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mRen2.Lewis rats Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R819 - R826. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. I. Gorodeski Estrogen Decrease in Tight Junctional Resistance Involves Matrix-Metalloproteinase-7-Mediated Remodeling of Occludin Endocrinology, January 1, 2007; 148(1): 218 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Monsalve, P. J. Oviedo, M. A. Garcia-Perez, J. J. Tarin, A. Cano, and C. Hermenegildo Estradiol counteracts oxidized LDL-induced asymmetric dimethylarginine production by cultured human endothelial cells Cardiovasc Res, January 1, 2007; 73(1): 66 - 72. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. X-D Song, P. Fan, W. Yue, Y. Chen, and R. J Santen Role of receptor complexes in the extranuclear actions of estrogen receptor {alpha} in breast cancer Endocr. Relat. Cancer, December 1, 2006; 13(Supplement_1): S3 - S13. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Castillo, G. Ceballos, D. Rodriguez, C. Villanueva, R. Medina, J. Lopez, E. Mendez, and E. F. Castillo Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1388 - C1394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Li, L. Zhou, and G. I. Gorodeski Estrogen Regulates Epithelial Cell Deformability by Modulation of Cortical Actomyosin through Phosphorylation of Nonmuscle Myosin Heavy-Chain II-B Filaments Endocrinology, November 1, 2006; 147(11): 5236 - 5248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Grassi, X. Fan, J. Rahnert, M. N. Weitzmann, R. Pacifici, M. S. Nanes, and J. Rubin Bone Re/Modeling Is More Dynamic in the Endothelial Nitric Oxide Synthase(-/-) Mouse Endocrinology, September 1, 2006; 147(9): 4392 - 4399. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, K. Chen, J. C. Shih, and C. T. Teng Estrogen-Related Receptors-Stimulated Monoamine Oxidase B Promoter Activity Is Down-Regulated by Estrogen Receptors Mol. Endocrinol., July 1, 2006; 20(7): 1547 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. X.-D. Song and R. J. Santen Membrane Initiated Estrogen Signaling in Breast Cancer Biol Reprod, July 1, 2006; 75(1): 9 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-b. Chen, S. Jia, A. G. King, A. Barker, S.-m. Li, E. Mata-Greenwood, J. Zheng, and R. R. Magness Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy Biol Reprod, May 1, 2006; 74(5): 832 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Almeida, L. Han, C. A. O'Brien, S. Kousteni, and S. C. Manolagas Classical Genotropic Versus Kinase-Initiated Regulation of Gene Transcription by the Estrogen Receptor {alpha} Endocrinology, April 1, 2006; 147(4): 1986 - 1996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mineo and P. W. Shaul Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae Cardiovasc Res, April 1, 2006; 70(1): 31 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Cruz, G. Douglas, J.-A Gustafsson, L. Poston, and K. Kublickiene Dilatory responses to estrogenic compounds in small femoral arteries of male and female estrogen receptor-{beta} knockout mice Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H823 - H829. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Wassmann, S. Wassmann, and G. Nickenig Progesterone Antagonizes the Vasoprotective Effect of Estrogen on Antioxidant Enzyme Expression and Function Circ. Res., November 11, 2005; 97(10): 1046 - 1054. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P Cooke and J. M Marshall Mechanisms of Raynaud's disease Vascular Medicine, November 1, 2005; 10(4): 293 - 307. [Abstract] [PDF]
|
|
|
|
 |
|
 A. Page, H. Reich, J. Zhou, V. Lai, D. C. Cattran, J. W. Scholey, and J. A. Miller Endothelial Nitric Oxide Synthase Gene/Gender Interactions and the Renal Hemodynamic Response to Angiotensin II J. Am. Soc. Nephrol., October 1, 2005; 16(10): 3053 - 3060. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Partovian, Z. Zhuang, K. Moodie, M. Lin, N. Ouchi, W. C. Sessa, K. Walsh, and M. Simons PKC{alpha} Activates eNOS and Increases Arterial Blood Flow In Vivo Circ. Res., September 2, 2005; 97(5): 482 - 487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V L Clifton, R Crompton, M A Read, P G Gibson, R Smith, and I M R Wright Microvascular effects of corticotropin-releasing hormone in human skin vary in relation to estrogen concentration during the menstrual cycle J. Endocrinol., July 1, 2005; 186(1): 69 - 76. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Schulz, E. Anter, M.-H. Zou, and J. F. Keaney Jr Estradiol-Mediated Endothelial Nitric Oxide Synthase Association With Heat Shock Protein 90 Requires Adenosine Monophosphate-Dependent Protein Kinase Circulation, June 28, 2005; 111(25): 3473 - 3480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Mendelsohn and R. H. Karas Molecular and Cellular Basis of Cardiovascular Gender Differences Science, June 10, 2005; 308(5728): 1583 - 1587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Guo, M. Razandi, A. Pedram, G. Kassab, and E. R. Levin Estrogen Induces Vascular Wall Dilation: MEDIATION THROUGH KINASE SIGNALING TO NITRIC OXIDE AND ESTROGEN RECEPTORS {alpha} AND {beta} J. Biol. Chem., May 20, 2005; 280(20): 19704 - 19710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jayachandran, R. Mukherjee, T. Steinkamp, P. LaBreche, M. P. Bracamonte, H. Okano, W. G. Owen, and V. M. Miller Differential effects of 17{beta}-estradiol, conjugated equine estrogen, and raloxifene on mRNA expression, aggregation, and secretion in platelets Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2355 - H2362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Gibson, L. Hahner, S. Osborne-Lawrence, Z. German, K. K. Wu, K. L. Chambliss, and P. W. Shaul Molecular Basis of Estrogen-Induced Cyclooxygenase Type 1 Upregulation in Endothelial Cells Circ. Res., March 18, 2005; 96(5): 518 - 525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Klinge, K. A. Blankenship, K. E. Risinger, S. Bhatnagar, E. L. Noisin, W. K. Sumanasekera, L. Zhao, D. M. Brey, and R. S. Keynton Resveratrol and Estradiol Rapidly Activate MAPK Signaling through Estrogen Receptors {alpha} and {beta} in Endothelial Cells J. Biol. Chem., March 4, 2005; 280(9): 7460 - 7468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. X. Liao, R. R. Magness, and D.-b. Chen Expression of Estrogen Receptors-{alpha} and -{beta} in the Pregnant Ovine Uterine Artery Endothelial Cells In Vivo and In Vitro Biol Reprod, March 1, 2005; 72(3): 530 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Chambliss, L. Simon, I. S. Yuhanna, C. Mineo, and P. W. Shaul Dissecting the Basis of Nongenomic Activation of Endothelial Nitric Oxide Synthase by Estradiol: Role of ER{alpha} Domains with Known Nuclear Functions Mol. Endocrinol., February 1, 2005; 19(2): 277 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hatzoglou, M. Kampa, C. Kogia, I. Charalampopoulos, P. A. Theodoropoulos, P. Anezinis, C. Dambaki, E. A. Papakonstanti, E. N. Stathopoulos, C. Stournaras, et al. Membrane Androgen Receptor Activation Induces Apoptotic Regression of Human Prostate Cancer Cells in Vitro and in Vivo J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 893 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Simoncini, P. Mannella, L. Fornari, A. Caruso, M. Y. Willis, S. Garibaldi, C. Baldacci, and A. R. Genazzani Differential Signal Transduction of Progesterone and Medroxyprogesterone Acetate in Human Endothelial Cells Endocrinology, December 1, 2004; 145(12): 5745 - 5756. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Chikani, W. Zhu, and E. J. Smart Lipids: potential regulators of nitric oxide generation Am J Physiol Endocrinol Metab, September 1, 2004; 287(3): E386 - E389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification Endocr. Rev., August 1, 2004; 25(4): 629 - 672. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. M. Miller, D. J. Tindall, and P. Y. Liu Of Mice, Men, and Hormones Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 995 - 997. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sengupta, S. Banerjee, N. K. Saxena, and S. K. Banerjee Thombospondin-1 Disrupts Estrogen-Induced Endothelial Cell Proliferation and Migration and Its Expression Is Suppressed by Estradiol Mol. Cancer Res., March 1, 2004; 2(3): 150 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-b. Chen, I. M. Bird, J. Zheng, and R. R. Magness Membrane Estrogen Receptor-Dependent Extracellular Signal-Regulated Kinase Pathway Mediates Acute Activation of Endothelial Nitric Oxide Synthase by Estrogen in Uterine Artery Endothelial Cells Endocrinology, January 1, 2004; 145(1): 113 - 125. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Mendelsohn and G. M.C. Rosano Hormonal Regulation of Normal Vascular Tone in Males Circ. Res., December 12, 2003; 93(12): 1142 - 1145. [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Farach-Carson and P. J. Davis Steroid Hormone Interactions with Target Cells: Cross Talk between Membrane and Nuclear Pathways J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 839 - 845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Qiu, M. A. Bosch, S. C. Tobias, D. K. Grandy, T. S. Scanlan, O. K. Ronnekleiv, and M. J. Kelly Rapid Signaling of Estrogen in Hypothalamic Neurons Involves a Novel G-Protein-Coupled Estrogen Receptor that Activates Protein Kinase C J. Neurosci., October 22, 2003; 23(29): 9529 - 9540. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Rosenfeld, C. Chen, T. Roy, and X.-T. Liu Estrogen Selectively Up-Regulates eNOS and nNOS in Reproductive Arteries By Transcriptional Mechanisms Reproductive Sciences, May 1, 2003; 10(4): 205 - 215. [Abstract] [PDF]
|
|
|
|
|
|
T. Simoncini, E. Rabkin, and J. K. Liao Molecular Basis of Cell Membrane Estrogen Receptor Interaction With Phosphatidylinositol 3-Kinase in Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2003; 23(2): 198 - 203. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, P < 0.05 vs. control cells; 
, P < 0.05 vs. sham. Similar findings were obtained in three independent experiments. B, Effect of ER
, P < 0.05 vs. sham. [Reprinted with permission from M. H. Wyckoff et al.: J Biol Chem 276:27071–27076, 2001 (