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American Journal of Physiology - Heart and Circulatory Physiology logoLink to American Journal of Physiology - Heart and Circulatory Physiology
. 2008 Jul 3;295(3):H1025–H1032. doi: 10.1152/ajpheart.00021.2008

Central estrogen inhibition of angiotensin II-induced hypertension in male mice and the role of reactive oxygen species

Baojian Xue 2,*, Yuanzi Zhao 2,*, Alan Kim Johnson 2,3,4,5, Meredith Hay 1,2
PMCID: PMC2544478  PMID: 18599599

Abstract

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP (Δ30.1 ± 2.5 mmHg). Either central infusion of Tempol or 17β-estradiol (E2) attenuated the pressor effect of ANG II (Δ10.9 ± 2.3 and Δ4.5 ± 1.4 mmHg), and the protective effect of E2 was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 (Δ23.6 ± 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central E2-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 ± 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (−1.8 ± 1.6% and −1.0 ± 1.8%). The ROS response to ANG II was also blocked by E2 (−3.2 ± 2.4%). The results suggest that the central actions of E2 are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production.

Keywords: sex hormone, blood pressure, oxidative stress, subfornical organ

it is generally accepted that the female sex hormone estrogen plays a major role in protecting premenopausal women from cardiovascular disease (15, 25, 44). A growing body of evidence indicates that the cardioprotective effects of estrogen may be related to its antioxidant properties. For example, in peripheral cardiovascular tissue, estrogen has been shown to reduce superoxide generation and ANG II-induced free radical production. Furthermore, estrogen exerts its antioxidative actions via stimulation of mitochondrial superoxide dismutase (MnSOD) and extracellular SOD (ecSOD) expression and activity, modulation of NADPH oxidase enzyme activity, and increased nitric oxide production (2, 5, 6, 11, 13, 21, 39, 43, 47). However, to date, there has been little investigation of the mechanisms underlying estrogen modulation of oxidative stress in brain cardiovascular regulatory regions.

ANG II, a peptide with potent vasoconstrictor actions, has been implicated in many forms of hypertension. In addition to its peripheral vasoconstrictor effects, ANG II is known to act at circumventricular organs (CVOs) to modulate autonomic control of blood pressure (BP) and heart rate (HR; Refs. 4, 8, 9, 30). As a CVO, the subfornical organ (SFO) lacks a blood-brain barrier and is thought to couple circulating signals such as ANG II with neural networks that mobilize effector systems (e.g., sympathetic outflow and vasopressin release) involved in maintaining BP and body fluid homeostasis (3, 10, 14). A growing body of evidence in recent years has implicated reactive oxygen species (ROS) as signaling intermediates in ANG II effects on BP regulation (22, 37, 50). Zimmerman and colleagues (52, 53) showed that hypertension induced by the systemic infusion of ANG II increased oxidative stress in SFO neurons. Adenoviral-mediated delivery of SOD targeted to the cytoplasm of SFO cells prevented ANG II-induced hypertension and increased ROS production. The inhibition of SFO NADPH oxidase, a key enzyme in free radical production, also prevented the expression of centrally mediated effects of ANG II (51). These results suggest that ANG II-induced ROS production in the central nervous system (CNS) is pivotal in ANG II-elicited BP and that the SFO is a key target of ANG II in the CNS.

Previous studies from our laboratory have shown that chronic ANG II infusion caused greater increases in BP in ovariectomized (OVX) wild-type (WT) female mice and in intact female mice treated intracerebroventricularly with a nonselective estrogen receptor (ER) antagonist ICI-182780 (ICI). The replacement of estrogen by a central infusion of 17β-estradiol (E2) attenuated the pressor effect of ANG II in OVX WT mice (48, 49). These findings suggest that female sex hormones, especially central estrogen, are critically involved in the protection against ANG II-dependent hypertension. However, the precise mechanisms responsible for the antihypertensive effect of estrogen and the central site of estrogen action are uncertain.

In addition to the expression of ANG II type 1 (AT1) receptors (29), SFO neurons also express ERs (36). Moreover, estrogen has been shown to decrease ANG II binding to AT1 receptors and to reduce the expression of AT1 receptors in SFO neurons (6, 16). Thus we hypothesized that estrogen may alter the physiological responses of SFO neurons to ANG II through interactions among mechanisms involving estrogen, ANG II, and oxidative stress. To explore this hypothesis, the present study first investigated the effects of the central administration of the SOD mimetic Tempol or estrogen on ANG II-induced hypertension in male mice. Second, living SFO slices and confocal microscopy were employed to determine whether estrogen inhibits ANG II activation of ROS production.

METHODS

All the experimental protocols in this study were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Iowa.

Surgical Procedures

Telemetry probe implantation.

Implantable mouse BP transmitters (TA11PA-C10, Data Sciences International, St. Paul, MN) were used to directly measure arterial pressure in individual animals. WT (C57BL6J) male mice (12–16 wk old) were anesthetized with a ketamine-xylazine mixture (100 and 10 mg/kg). The carotid artery of the mouse was accessed with a ventral midline incision. The left carotid artery was isolated, and the catheter of a telemetry probe was inserted into the vessel. Through the same ventral incision, a subcutaneous tunnel was formed across the right pectoral area and was enlarged to form a pocket along the right flank. The body of the transmitter was slipped into the pocket and secured with tissue adhesive. The ventral incision was then closed with suture.

Chronic intracerebroventricular cannula implantation.

After baseline BP and HR recordings were obtained, the mice were again anesthetized with a ketamine-xylazine mixture, and an intracerebroventricular cannula with an osmotic pump (Alzet Brain Infusion Kits, Alzet) was implanted into the right lateral ventricle (at the following coordinates: 0.3 mm caudal, 1.0 mm lateral to bregma, and 3.0 mm below the skull surface) for chronic infusions of Tempol (10 ng·kg−1·day−1, Sigma), E2 (30 μg·kg−1·day−1, Sigma), or E2 + ICI (1.5 μg·kg−1·day−1, a nonselective ER antagonist, Tocris). At the end of the experiment, the animals were euthanized and perfused transcardially with saline followed by fixative. The location of the lateral ventricle cannula implantation was verified histologically.

Osmotic pump implantation.

The mice were anesthetized with inhalational isoflurane to allow for the implantation of osmotic pumps. Osmotic pumps (model 1002, Alzet) containing ANG II (Sigma Chemical) at a concentration sufficient to allow an infusion rate of 800 ng·kg−1·min−1 were implanted subcutaneously on the left side of the back.

SFO brain slice preparation.

SFO brain slices were obtained from adult male (8–10 wk old) mice. The brain slice dissection was performed according to an established protocol. Briefly, under halothane anesthesia the mice were decapitated and the forebrain was rapidly removed and immersed in ice-cold cutting solution containing (in mM) 220 sucrose, 3 KCl, 1.25 NaH2PO4, 6 MgSO4, 26 NaHCO3, 0.2 CaCl2, 10 glucose, and 0.43 ketamine. After 1 to 2 min, the forebrain was trimmed and glued onto the block of a vibrotome, and 150-μm-thick coronal forebrain slices were then cut at the level of the SFO in ice-cold, oxygenated cutting solution. The slices were placed in oxygenated artificial cerebrospinal fluid (aCSF) 60 min before incubation in dihydroethidium (DHE, 20 μM, Invitrogen).

Experimental Protocol

Measurement of BP and HR.

All mice were allowed 7 days to recover from transmitter implantation surgery before any measurements were made. Thereafter, BP and HR were telemetrically recorded and stored with the Dataquest ART data acquisition system (Data Sciences International).

To determine the effects of Tempol, E2, or E2 + ICI on ANG II-induced hypertension in WT mice, intracerebroventricular cannulas with osmotic pumps were implanted into the right lateral ventricle for chronic infusions of vehicle, Tempol, E2, or E2 + ICI for 14 days. On day 7, osmotic pumps filled with ANG II were implanted subcutaneously.

Evaluation of BP responses to autonomic blockade.

BP levels in males were also measured in the presence of the ganglionic blocker hexamethonium (30 mg/kg ip). Ganglionic blockade was repeated two times in each animal, during baseline and after 7 days of ANG II infusion. On the day of ganglionic blockade experiments, the mice were allowed to stabilize for at least 60 min, after which time BP was recorded for 20 min before and after hexamethonium injection.

Measurement of intracellular ROS production in SFO brain slices.

ROS generation was studied using real-time fluorescence microscopy with the measurement of conversion of the fluorescent probe DHE to ethidium. DHE is a cell permeable indicator that reacts with superoxide radicals to form ethidium that in turn intercalates with DNA and provides nuclear fluorescence at an excitation wavelength of 520 nm and emission wavelength of 610 nm. Ethidium fluorescence was detected using an Olympus FV500 confocal system equipped with a green HeNe laser for excitation and a long-pass red filter. SFO brain slices were incubated in DHE for 45 min at 34°C and then mounted in an imaging chamber perfused with oxygenated aCSF. Ethidium fluorescence intensity was recorded every 30 s for 6 min.

To determine the effect of ANG II on ROS production, basal production of ROS was first measured. The perfusate was then switched to ANG II. Ethidium intensity was also recorded during the superfusion of the slices with ANG II plus irbesartan to determine whether the effect of ANG II on the generation of ROS was mediated by AT1 receptors. To confirm that ANG II-mediated increases in ethidium fluorescence were due to superoxide anion production, responses to ANG II were tested in slices preincubated for 45 min in Tempol together with DHE. To determine the effects of E2, ICI, and E2 + ICI on ANG II-evoked generation of ROS in SFO neurons, the slices were preincubated in E2, ICI, or both together with DHE for 45 min before testing the responses to ANG II, respectively.

Data Analysis

Imaging data were generated from an Olympus FV500 microscope and then analyzed by Image Pro Plus. Real-time ethidium fluorescence intensity was recorded every 30 s for 6 min during the superfusion of aCSF or drugs. Twenty-four absolute values of ethidium fluorescence intensity were obtained corresponding to each time point. The first twelve absolute values of ethidium fluorescence during superfusion of aCSF were averaged, and this value was used as a baseline intensity, and the next twelve values during drug superfusion were compared with the baseline to get a percent change of ethidium intensity. Finally, the data are expressed as the averaged percent change of fluorescence measured every 30 s for 6 min of the baseline recording and 6 min of the experimental recording.

Mean arterial pressure (MAP) and HR collected for 4 and 14 consecutive days before and during Tempol, E2, and ANG II pump implantation, respectively, were plotted as mean values.

All data are expressed as means ± SE. Statistical analyses of the effects of the central administration of Tempol, E2, or E2 + ICI on BP before and after ANG II infusion were performed with two-way ANOVA for repeated measures (Sigma Stat, version 2.06). Post hoc analysis was performed with Fisher least significant difference multiple comparison test where appropriate. A one-way ANOVA was used for comparing changes in BP and ethidium fluorescence intensity. Statistical significance was accepted at P < 0.05.

RESULTS

Effects of Lateral Ventricle Infusion of Tempol, E2, or E2 + ICI on ANG II-Induced Hypertension in Male Mice

Baseline MAP in males was unaltered during the central infusion of either Tempol (10 ng·kg−1·day−1, Fig. 1A), E2 (30 μg·kg−1·day−1, Fig. 2A) or E2 + ICI (1.5 μg·kg−1·day−1, Fig. 2A). Central Tempol (n = 6, Fig. 1, A and B) significantly inhibited and E2 (n = 6, Fig. 2, A and B) prevented the increase in MAP induced by ANG II (Δ10.9 ± 2.3 and Δ4.5 ± 1.4 mmHg, respectively, P < 0.05) compared with that seen in mice with central vehicle plus systemic ANG II (Δ30.1 ± 2.5 mmHg, n = 6). The concurrent administration of ICI prevented the protective effect of E2 (Δ23.6 ± 3.1 mmHg, n = 6). ANG II infusion did not change HR in any group (Figs. 1C and 2C).

Fig. 1.

Fig. 1.

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Effect of chronic intracerebroventricular infusion of Tempol on ANG II-induced hypertension. Daily measurement of mean arterial pressures (MAPs, A) and heart rate (HR, C) before and during systemic infusion of ANG II in male mice with intracerebroventricular infusion of vehicle or Tempol. B: increases in MAP averaged across days induced by ANG II infusion in central vehicle or Tempol-treated mice. C, control day (followed by 14 days of central vehicle or Tempol and 7 days of ANG II infusion). *P < 0.05 compared with baseline; #P < 0.05 compared with vehicle + ANG II mice.

Fig. 2.

Fig. 2.

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Effect of chronic intracerebroventricular infusion of estrogen and estrogen receptor antagonist on ANG II-induced hypertension. Daily measurement of MAP (A) and HRs (C) before and during systemic infusion ANG II in intracerebroventricular vehicle-, 17β-estradiol (E2)-, or E2 + ICI-182780 (ICI)-treated male mice. B: increases in MAP across days induced by ANG II infusion in all groups of male mice. Control days are followed by 14 days of vehicle, E2, or E2 + ICI and 7 days of ANG II infusion. #P < 0.05 compared with males given central E2.

Effects of Autonomic Blockade on BP

Figure 3 shows decreases in BP with acute ganglionic blockade in all groups of males. The averaged reduction in the BP response to hexamethonium injection before infusion of ANG II was −26.9 ± 3.7 mmHg. Following 7 days of ANG II infusion, an acute hexamethonium injection resulted in a greater reduction in BP in central E2 + ICI-treated (−50.2 ± 5.3 mmHg) and central vehicle-treated (−61.0 ± 5.8 mmHg) males compared with central Tempol (−30.5 ± 4.4, P < 0.05)- or with central E2-treated males (−27.0 ± 4.4 mmHg, P < 0.05). The results suggest that the attenuated effects of ANG II on BP in males treated centrally with Tempol or estrogen involve a decrease in sympathetic outflow.

Fig. 3.

Fig. 3.

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Effect of chronic intracerebroventricular infusion of E2, E2 + ICI, or Tempol on MAP in response to ganglionic blockade with hexamethonium before beginning ANG II infusions (control) and on day 7 of ANG II infusion. #P < 0.05 compared with control, central Tempol-, or E2-treated male mice.

Effects of ANG II on Intracellular Generation of ROS

Figure 4A shows the representative bright field and fluorescent images of a SFO brain slice loaded with DHE (20 μM). The intensity of ethidium fluorescence peaked after a 30-s superfusion of brain slices with ANG II (100 nM). The average percent change in ethidium intensity during superfusion with aCSF alone or aCSF containing ANG II for 6 min is shown in Fig. 4B. The application of aCSF alone had no significant effect on ethidium intensity (0.8 ± 1.2%, n = 7, P > 0.05). Superfusion with ANG II increased ethidium intensity in SFO neurons by 21.5 ± 2.5% (n = 18, P < 0.05). In the presence of an AT1 receptor blocker, irbesartan (1 μM), ANG II did not evoke any changes in ethidium intensity (−1.8 ± 1.6%, n = 7, P < 0.05), suggesting that the ANG II-induced increase in intracellular ROS production is mediated by AT1 receptors (Fig. 4C).

Fig. 4.

Fig. 4.

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A: representative confocal bright field images of a subfornical organ (SFO) brain slice loaded with dihydroethidium and the significant increases in ethidium intensity after 30 s of superfusion of brain slices with ANG II. B: percent changes of ethidium intensity over time before and after an application of ANG II to the bath solution. C: average percent change of ethidium intensity measured every 30 s for 6 min in SFO neurons following superfusion of ANG II, artificial cerebrospinal fluid (aCSF), and ANG II + irbesartan. *P < 0.05 compared with aCSF or ANG II + irbesartan.

Effect of Tempol on ANG II-Induced Intracellular ROS Production

SFO brain slices were preincubated in Tempol (5 mM) together with DHE for 45 min and then changes in fluorescence intensity were recorded during the superfusion of the slices with Tempol or Tempol plus ANG II (100 nM). Tempol significantly attenuated ANG II-induced increases in ethidium fluorescence intensity (−1.0 ± 1.8%, n = 7, P < 0.05; Fig. 5).

Fig. 5.

Fig. 5.

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Effect of Tempol on ANG II-induced reactive oxygen species (ROS) production. The average percent change of ethidium intensity measured every 30 s for 6 min in SFO neurons following superfusion of brain slices with ANG II, Tempol, and ANG II + Tempol is shown. *P < 0.05 compared with Tempol or ANG II + Tempol.

Effects of E2 on ANG II-Induced Intracellular ROS Production

In the brain slices incubated with DHE, an acute application of E2 (100 nM) did not result in a significant inhibition of ROS production (−3.2 ± 0.9%, n = 8, P > 0.05). However, in the brain slices preincubated with E2 (100 nM), ANG II-induced ROS production was significantly attenuated (−3.2 ± 2.4%, n = 8, P < 0.05; Fig. 6).

Fig. 6.

Fig. 6.

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Effect of E2 on ANG II-induced ROS production. The average percent change of ethidium intensity measured every 30 s for 6 min in SFO neurons following superfusion of brain slices with ANG II, E2, and ANG II + E2 is shown. *P < 0.05 compared with E2 or ANG II + E2.

Effect of ICI on E2 Modulation of ANG II-Induced ROS Production

Brain slices were preincubated in E2 (100 nM) and ICI (10 μM) together with DHE for 45 min before testing for responses to ANG II. In the presence of E2 and ICI, the ANG II-induced increase in ROS production (16.4 ± 3.1%, n = 6) was much greater than that evoked in the presence of E2 alone (−3.2 ± 2.4%, n = 8, P < 0.05). Preincubation with ICI alone had no effect on ANG II-induced increases in ROS production in SFO neurons (20.3 ± 4.2%, n = 8). The data suggest that the inhibitory effect of E2 on the ANG II-evoked generation of ROS in SFO neurons requires the activation of ERs (Fig. 7).

Fig. 7.

Fig. 7.

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Effect of ICI on E2 modulation of ANG II-induced ROS production. The average percent change of ethidium intensity measured every 30 s for 6 min during the perfusion of the slices with ANG II, E2 + ICI, ICI alone, ANG II + ICI, and ANG II + E2 + ICI. *P < 0.05 compared with E2 + ICI or ICI alone.

DISCUSSION

The main findings of this study are as follows: 1) the ANG II-induced increases in BP in male mice are inhibited by the central administration of Tempol, 2) the central activation of ERs in male mice inhibits ANG II-induced increases in sympathetic outflow and hypertension, 3) ANG II and AT1 receptor activation increases ROS in living SFO brain slices, and 4) ER activation in the SFO inhibits ANG II-induced increases in ROS. These observations suggest that estrogen inhibits ANG II-induced hypertension and activation of SFO neurons via interactions with intracellular ROS production.

The capacity of DHE to detect superoxide anion production when combined with confocal imaging to eliminate out-of-focus light allows the monitoring of ROS formation and change. Zimmerman et al. (52, 53) reported that CVO cells in primary culture incubated with ANG II or frozen SFO brain slices derived from mice after long-term peripheral ANG II infusions show marked increases in DHE fluorescence. The present study further demonstrated that ANG II rapidly increases ethidium intensity in the living SFO brain slices. When compared with other reports (45, 53), we used a concentration of ANG II (100 nM) within the physiological range (20) and obtained significant ROS responses, suggesting that the living brain slice is more sensitive to ANG II compared with cultured neurons. The time course of ANG II-induced increases in ROS examined in cultured aortic vascular smooth muscle cells (VSMCs) with dichlorofluorescein fluorescence showed that ANG II causes an abrupt increase in ROS generation within 5–10 s (33, 38). Although the latency for the onset of ROS generation was not precisely determined in the present study, the DHE fluorescence response to ANG II stimulation peaked at 30 s, followed by a plateau for the remainder of the 6-min recording period. These data provide additional evidence demonstrating that ANG II rapidly stimulates the generation of ROS in brain tissues.

Tempol is a membrane-permeable, metal-independent SOD mimetic that has been shown to be specific for the superoxide anion and to effectively scavenge and chemically reduce the superoxide anion (26). Nishiyama and colleagues (27) have demonstrated that intravenous administration of Tempol normalizes vascular superoxide production and decreases MAP in ANG II-infused rats. Lu et al. (23) reported that intracerebroventricular Tempol administration influences central sympathetic neural circuits in a dose-dependent manner and attenuates renal sympathetic nerve discharge to central ANG II infusion. Recent studies by Zimmerman et al. (52, 53) further demonstrate that the overexpression of SOD in the SFO eliminates MAP, HR, and dipsogenic responses to systemic or intracerebroventricular administration of ANG II. Consistent with results from these previous studies, the present study demonstrated that intracerebroventricular administration of Tempol results in a significant decrease in MAP and in sympathetic outflow induced by the systemic infusions of ANG II in mice. Furthermore, the preincubation of Tempol blocked ANG II-induced increases in fluorescence intensity in SFO slices, suggesting that ANG II-mediated superoxide anion production is the main source for the induced increases in ethidium fluorescence. These results confirm and provide insights into the key mechanisms by which ANG II influences BP and sympathetic outflow through its capacity to produce ROS. In addition, the SFO is dense with AT1 receptors and is thought to be pivotal for long-term systemic infusion of ANG II-elicited BP and dipsogenic actions (52, 53). Considerable evidence has shown further that the cellular and functional effects of ANG II are mediated by its interaction with membrane AT1 receptors (42). Therefore, our observation that irbesartan, an AT1 receptor antagonist, blocked the increases in ethidium fluorescence intensity induced by ANG II in SFO brain slices was not unexpected.

We have previously reported that OVX facilitates the development of ANG II-dependent hypertension, suggesting that estrogen has a protective role against this form of high blood pressure in female mice (48). Furthermore, in OVX WT mice, the central infusion of E2 attenuated the pressor effect of ANG II, and this attenuated effect of ANG II on the BP response involves a decrease in sympathetic outflow (49). However, the effect of central ER activation on ANG II-induced hypertension in males has not been previously reported. It has been shown that endogenous production of estrogen in men plays a significant role in cardiovascular health and that estrogen acts on the male cardiovascular system in a manner similar to that in women (41). For example, an acute intravenous administration of E2 in men improves coronary blood flow and cutaneous vasodilator responses to acetylcholine (1, 17) and abolishes abnormal coronary vasoconstriction in response to an exogenous cold stimulus (31, 32). Chronic low-dose estrogen supplementation in hypogonadal men attenuates vasoconstrictor responses to norepinephrine and ANG II (19), increases basal nitric oxide release in forearm resistance arteries, and decreases baseline and stress-induced increases in blood pressure, which is consistent with previous reports in perimenopausal women (18, 40). Therefore, the recognition that high doses of estrogen may produce effects distinct from those obtained at physiological levels and an appreciation of the complexity of the relationship between sex steroids and the cardiovascular system in women suggest a need for further studies to clarify the roles of these hormones in men. The present study is the first to show that in male mice, central E2 inhibits ANG II-induced hypertension and sympathoexcitation and that this is associated with the blockade of the effects of ANG II generation of intracellular ROS in SFO neurons. The results provide new evidence that activation of central ERs can inhibit central ANG II actions via an interaction with ROS production.

The effects of estrogen on ROS production have been investigated in studies in cultured VSMCs (21, 39), endothelial cells (13, 43, 47), cardiac fibroblasts (2), and isolated blood vessels (5, 11). In these studies, OVX female animals were treated with E2 for 2–4 wk before samples were isolated or the cultured cells were directly incubated with E2 for 12–48 h. These investigators found that estrogen exerts radical-scavenging effects through increased nitric oxide production, stimulation of MnSOD and ecSOD expression and activity, and modulation of NADPH oxidase expression and activity, which ultimately lead to decreased ROS production. In the present study, an acute application of E2 had no effect on ROS production in the living SFO brain slice, but a preincubation with E2 did prevent ANG II-induced ROS generation. The inhibitory effect of E2 on ANG II-induced activation of SFO neurons is consistent with our previous studies in area postrema (AP) neurons (28). Besides the SFO and AP, there are additional central sites where E2 could act to attenuate the actions of ANG II. For example, neurons of the hypothalamic paraventricular nucleus (PVN) and the rostral ventrolateral medulla (RVLM) are known to be involved in the regulation of autonomic activity, and both nuclei express AT1 and ERs. The activation of ERs has been shown to inhibit PVN (12) and RVLM (46) neuronal activity, and PVN glutamate-induced increases in BP (12).

It has been established that the effects of E2 on the cardiovascular system may be mediated, at least in part, by its modulation of the renin-angiotensin system. OVX alone increases AT1 receptor mRNA, AT1 receptor binding density, and angiotensin-converting enzyme (ACE) activity in the aorta (11), adrenal cortex (16), SFO, and PVN (6, 16). These effects are prevented by the replacement of E2 at physiological levels. A superphysiological dose of E2 reverses the increases in ACE and AT1 receptors in the SFO and the PVN to decreases (6). These results suggest that E2 may alter the production of and the responsiveness to ANG II. Extensive work has identified ROS as novel molecules implicated in the intracellular signaling mechanisms of ANG II and NADPH oxidase as the primary source of superoxide anion. In cultured rat cardiac fibroblasts, VSMCs, and endothelial cells, E2 is reported to completely prevent ANG II-induced NADPH oxidase activity and ROS production (2, 13, 39). Thus it is reasonable to speculate that in the present study E2 blockade of ANG II-induced ROS production is related to the inhibitory effects of E2 on ANG II-induced NADPH oxidase activity.

Physiologically relevant concentrations of estrogen have both rapid and long-term positive cardiovascular effects that are mediated by both ER-α and ER-β (24). Interestingly, our in vivo and in vitro results both showed that although ICI, a nonselective ER antagonist, significantly inhibits the effects of E2 on ANG II-induced increases in BP and ROS production in SFO neurons, the inhibitory effect of ICI on E2 was not complete. It has been shown that the effect of E2 to prevent ANG II-mediated responses in endothelial cells may also involve nonspecific effects (i.e., receptor independent), such as altering physiochemical membrane properties (7, 13). Perhaps E2 also acts as an antioxidant independent of a receptor-mediated mechanism (34, 35). Thus receptor-independent actions of E2 may play a partial role in the antioxidant effect of the steroid in the present study.

In summary, our data demonstrate the interactions between E2 and ANG II and their role in oxidative stress in SFO neurons. The present study suggests that estrogen inhibits ANG II-induced hypertension and activation of SFO neurons via interactions with intracellular ROS production.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-62261, HL-59676, and HL-14388.

Acknowledgments

Present address of Y. Zhao: Div. of Neonatology and Developmental Biology, Dept. of Pediatrics, David Geffen School of Medicine, Univ. of California at Los Angeles, Los Angeles, CA. Present address of M. Hay: The Univ. of Arizona, Tucson, AZ.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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