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17-Estradiol inhibits angiotensin II activation of area postrema neurons

¹Dalton Cardiovascular Research Center, ²Department of Veterinary Biomedical Sciences, and ³National Center for Gender Physiology, University of Missouri, Columbia, Missouri 65211

Submitted 23 February 2003 ; accepted in final form 18 June 2003

	ABSTRACT

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It is well established that the area postrema, as a circumventricular organ, is susceptible to modulation by circulating hormones and peptides. Furthermore, activation of the area postrema has been shown to modulate central neurons involved in the regulation of cardiovascular function and blood pressure. In particular, the vasoactive peptide angiotensin II (ANG II) has been shown to inhibit baroreflex regulation of heart rate and increase sympathetic outflow and blood pressure via activation of area postrema neurons. Estrogen is thought to protect against hypertension in both humans and animal models and has been shown in a number of systems to alter the effects of ANG II. The purpose of the present study was to determine the effects of estrogen on ANG II activation of area postrema neurons. In this study, the effects of ANG II and KCl on fura 2-measured cytosolic Ca²⁺ concentration ([Ca²⁺]_i) responses in cultured area postrema neurons in the presence and absence of 12-h exposure to 100 nM 17-estradiol (E₂) were evaluated. In neurons incubated in control vehicle media, 50 nM ANG II increased [Ca²⁺]_i by 92 ± 12%. In neurons preincubated with 100 nM E₂, ANG II increased [Ca²⁺]_i by only 68 ± 11%, for a total inhibition of the ANG II-evoked response of 24%. Coapplication of the estrogen receptor antagonist ICI-182,780 did not inhibit the effects of E₂. In the same cells in which the effects of E₂ on ANG II-evoked responses were tested, the effects of incubation in E on the depolarization-induced increased [Ca²⁺²]_i due to 60 mM KCl were also tested. Incubation of the cells with 100 nM E increased the KCl-evoked [Ca²⁺²]_i response, and this response was blocked by ICI-182,780. These results suggest that in the area postrema, estrogen may utilize multiple pathways to modulate neural activity and responses to ANG II.

circumventricular organs; estrogen; vasoactive peptides; fura #2 imaging

The area postrema is a circumventricular organ in the medulla that is subject to modulation by both neural and humoral factors. Chemical activation of the area postrema has been shown to be involved in a number of physiological functions including taste sensitivity, emesis, and cardiovascular regulation (7, 16). Some peptide hormones are believed to exert their central effects primarily through actions at neurons within the area postrema. Neurons within the area postrema are known to be activated and express receptors for a number of different neuropeptides, including ANG II. Substantial evidence has been accumulated suggesting that circulating vasoactive peptides such as ANG II act at the area postrema to modulate sympathetic and vagal activity and ultimately the baroreflex regulation of blood pressure (8, 9, 25, 29, 33). Estrogen receptors (ERs) are also known to be highly expressed in the area postrema, and both the ER and the ER receptor subtypes have been identified within the area postrema (21, 32, 43).

The purpose of the present study was to 1) determine the effects of 17-estradiol (E₂) on ANG II activation of area postrema neurons and 2) compare these effects with the effects of E₂ on depolarization-induced activation of area postrema neurons.

	METHODS

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Neuronal culture. The experimental protocols in this study were reviewed and approved by the Animal Care and Use Committee of the University of Missouri.

Primary cultures of neurons were prepared from the area postrema of 10- to 16-day-old male and female postnatal rats. The dissociation protocol was done according to established procedures (1, 14). Briefly, rats were anesthetized with halothane, and the hindbrain and cerebellum were rapidly removed and placed in 4°C physiological buffer. A 500-µm-thick horizontal medullary slice, including the area postrema, was obtained using a vibratome. Under a dissection microscope, the area postrema was identified by its distinguishing shallow orange color and cut away from the surrounding tissue. The tissue was then incubated for 20 min in Earle's balanced salt solution (Sigma) containing 5 mg/ml papain, 123 nM cysteine, 0.5 mM EDTA, and 1.5 mM CaCl₂ maintained at 37°C. The tissue was then triturated in a papain-free solution with serially smaller pipettes until most of the tissue was dissociated. Dissociated cells were rinsed in DMEM (Sigma) and finally plated on poly-L-lysine-coated coverslips. Cells grown in minimum essential media containing 10% normal fetal bovine serum (FBS; GIBCO-BRL) were used in the initial experiments testing intracellular Ca²⁺ concentration ([Ca²⁺]_i) responses to KCl and different doses of ANG II. In experiments studying the modulation of ANG II- and KCl-evoked responses by E₂, the media used were phenol free and supplemented with 10% dextran-stripped FBS (GIBCO-BRL). Cells were maintained in minimum essential media containing 8 ng/ml nerve growth factor at 37°C in a CO₂ incubator for 6 days before being imaged. On an average, area postrema harvested from seven rats were plated on to 10 glass coverslips.

Calcium measurements. Changes in [Ca²⁺]_i were assessed by ratiometric imaging of the calcium indicator dye fura 2 as described previously (14). The day before the experimental protocols were begun, the neurons were incubated for 12 h in media either 1) free of E₂, 2) with 100 nM E₂, or 3) with 100 nM E₂ plus 10 µM of the selective ER antagonist ICI-182,780 (ICI). Similar doses of E₂ and ICI have been reported in literature (4, 5, 31, 36, 39, 40). Neurons were then loaded with fura 2 ratiometric dye by the addition of 4 µM fura 2-AM (Molecular Probes) to the culture media and incubated at 37°C in 5% CO₂. After 30 min, the cultures were rinsed once with balanced salt solution containing (in mM) 139 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.1 MgSO₄, 0.9 NaH₂PO₄, 27.75 glucose, and 10 HEPES. The coverslip was then placed in the recording chamber, which was fixed to the stage of an Olympus inverted fluorescent microscope equipped with a 100-W Hg fluorescent lamp, an Olympus x40 oil objective, a 410-nm dichroic mirror, a 520-nm high-pass emission filter, and a computer-controlled shutter and filter wheel with 340- and 380-nm excitation filters. With this configuration, three to four neurons per coverslip were used for measurement within a given visual field. Fluorescent images of the cells were obtained using a Sensys digital camera (Photometrics). Paired images were collected for 1.5 s and analyzed using an image processor (Axon Imaging Workshop). Ratio measurements were calculated every 10 s according to the method of Grynkiewicz and colleagues (11).

Data analysis. Values are means ± SE from cell groups. Means were compared by ANOVA, followed by Fisher's least-significant-difference test, and differences at P < 0.05 were considered statistically significant (SigmaStat version 2.3). An unpaired t-test was used for the data shown in Fig. 2B.

View larger version (16K): [in this window] [in a new window]   Fig. 2. A: average increases in [Ca2+]i with increasing concentrations of ANG II (5–500 nM) in cultured area postrema neurons grown in media containing normal serum. Area postrema neuronal [Ca2+]i increased in a dose-dependent manner, and similar responses were obtained when the doses were randomized (n = 12). *P < 0.005, significantly different from 5.0 and 50 nM ANG II doses (ANOVA). B: comparison of average increases in [Ca2+]i with 50 and 500 nM concentrations of ANG II in cultured area postrema neurons grown in media containing normal serum (n = 12) or dextran-stripped serum (n = 10). *P < 0.05 (unpaired t-test).

	RESULTS

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All experiments were performed on area postrema neurons maintained in culture for 6 days. The distinct morphology of area postrema neurons after 6 days in culture, including phase-bright, 8- to 10-µm-diameter soma and 2- to 5-µm-long thin processes, were readily distinguishable from the flat, large glial cells.

In all experiments, cells were tested with both ANG II and a general depolarization stimulus of KCl (60 mM). Figure 1, A and B, shows typical [Ca²⁺]_i responses of an area postrema neuron to ANG II (50 nM) and KCl (60 mM) in cultures grown in media containing normal FBS and phenol-free media supplemented with dextran-stripped FBS, respectively. The response to 50 nM ANG II reached a maximum within 10 s and recovered to baseline within 2 min. The response to 60 mM KCl was similar in magnitude to the ANG II response but recovered back to baseline within 30 s. The activation of area postrema neurons by both ANG II and KCl was repeatable and reversible. However, in neurons grown in media containing dextran-stripped FBS, the response to ANG II (50 nM) were higher and KCl responses were lower. The recovery to baseline was also slower in neurons grown in media containing dextran-stripped FBS.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Representative traces of ANG II (50 nM)- and KCl (60 mM)-evoked increases in intracellular Ca2+ concentration ([Ca2+]i) in cultured area postrema neurons grown in media containing normal serum (A) or dextran-stripped serum (B). The magnitude of responses to ANG II was higher and to KCl was lower in neurons grown in dextran-stripped media. Recovery to baseline values was slower after ANG II (2 min) than KCl (30 s) application.

The effects of ANG II on area postrema neuronal [Ca²⁺]_i were increased with increasing concentrations of ANG II in neurons grown in normal serum. Figure 2 illustrates the averaged effects of increasing ANG II concentrations in normal serum. Similar responses were obtained when the doses were applied randomly. Responses to 50 and 500 nM doses of ANG II were also tested in neurons grown in media containing dextran-stripped serum. In this cell culture, calcium responses to ANG II peaked at 50 nM (92 ± 12%), and 500 nM ANG II produced marginally higher increases in intracellular calcium (98 ± 3%).

To determine whether E₂ can modulate the ANG II-induced responses in area postrema neurons, neuronal cultures grown in media containing dextran-stripped serum were incubated in 100 nM E₂ for 12 h before being loaded with fura 2. Incubation in 100 nM E₂ had no apparent effect on the resting baseline [Ca²⁺]_i. Figure 3 illustrates the averaged effects of E₂ and the specific ER antagonist ICI on ANG II-induced increases in [Ca²⁺]_i. In these experiments, cell cultures were incubated in either control vehicle media or media with 100 nM E₂. Incubation with 100 nM E₂ inhibited the ANG II-evoked response. In neurons incubated in control vehicle media, 50 nM ANG II increased [Ca²⁺]_i by 92 ± 12%. In neurons preincubated with 50 nM E₂, ANG II increased [Ca²⁺]_i by only 68 ± 11%, for a total inhibition of the ANG II-evoked response of 24%. To determine ER selectivity, the effects of E₂ on ANG II-evoked responses were tested in the presence of the ER receptor antagonist ICI. In these experiments, ICI was added concomitantly with E₂ during the preincubation period. The inhibitory effect of E₂ on the ANG II-evoked response was not blocked by the ER receptor antagonist ICI. Importantly, when added alone, in the absence of E₂, ICI (10 µM) exhibited partial agonist effects and inhibited the ANG II-evoked response to the same extent as did E₂.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Preincubation of area postrema neurons in 100 nM 17-estradiol (E2; 17-E2) for 12 h inhibited ANG II-evoked increases in [Ca2+]i. The inhibitory effect of E2 on the ANG II (50 nM) responses was not blocked by the estrogen receptor (ER) antagonist ICI-182,780 (ICI). ICI (10 µM) alone, in the absence of E2, exhibited partial agonist effects and inhibited the ANG II-evoked response to the same extent as E2. *P < 0.05.

In the same cells in which the effects of E₂ on ANG II-evoked responses were tested, we also tested the effects of incubation in E₂ on the depolarization effects due to 60 mM KCl. Incubation of the cells with 100 nM E increased the KCl-evoked [Ca²⁺²]_i response (Fig. 4). In neurons incubated in control vehicle media, 60 mM KCl increased [Ca²⁺]_i by 45 ± 6%. In neurons preincubated with 100 nM E₂, 60 mM KCl increased [Ca²⁺]_i by 63 ± 9%, for a total increase in the KCl response of 23 ± 8%. This effect of E₂ was totally blocked by the addition of the ER receptor antagonist ICI during the preincubation period. Furthermore, the ICI compound exhibited no partial agonist effects and had no effect on the KCl response when added in the absence of E₂.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Preincubation of area postrema neurons in 100 nM E2 for 12 h facilitates KCl-induced increases in [Ca2+]i. This effect is totally blocked by the ER antagonist ICI (10 µM) during the preincubation period, and ICI alone did not alter the KCl response. *P < 0.05 (ANOVA); P < 0.05, significantly smaller compared with control KCl responses.

	DISCUSSION

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This study is the first to report on the effects of E₂ on ANG II- and KCl-induced activation of area postrema neurons. Twelve-hour exposure with estradiol effectively attenuated ANG II activation of area postrema neurons but facilitated KCl-induced activation of area postrema neurons. Importantly, the ability of estradiol to attenuate the effects of ANG II on area postrema [Ca²⁺]_i was not blocked by the selective ER antagonist ICI. Furthermore, ICI, when applied alone, had no effect on [Ca²⁺]_i; when coapplied with ANG II, ICI mimicked the effects of E₂, suggesting that ICI has partial agonist effects on the ANG II-induced increase of [Ca²⁺]_i. However, ICI did effectively block E₂'s facilitation of KCl-induced activation. These results provide new insights as to the role of E₂ in the regulation of area postrema neuronal activity and suggest that within the same neuron, E₂ may activate multiple ERs or multiple signaling pathways.

ANG II is thought to mediate its cellular and functional effects via a wide variety of ANG II receptor subtype 1 (AT₁) signaling pathways in peripheral tissue as well as central neurons. Anatomic and functional studies have confirmed the presence of AT₁ receptors in the area postrema of neonatal rats (30). A commonly described AT₁ receptor signaling pathway is G protein-mediated stimulation of phospholipase C and phosphoinositide hydrolysis with subsequent calcium mobilization. This increase in intracellular calcium causes activation of Ca²⁺-dependent enzymes, such as PKC and Ca²⁺/calmodulin-dependent protein kinase II (46, 47). Typically, ANG II causes an early spike response in [Ca²⁺]_i, followed by a slow return to resting levels. ANG II-induced [Ca²⁺]_i spike elevation is predominantly dependent on the release of calcium from intracellular stores by inositol-1,4,5-trisphosphate and independent of voltage-gated calcium currents. The slow return to baseline is partly attributed to calcium entry via voltage-gated calcium channels after the initial spike (45). Alterations in AT₁ receptor expression or any part of the signaling pathways could potentially affect ANG II-mediated [Ca²⁺]_i responses. Estrogen-mediated decreases in AT₁ receptor expression have been shown in in vitro cultures of vascular smooth cells (28). Although the effects of estrogen in vitro on neuronal AT₁ receptor expression are not known, several studies have shown an alteration in ANG II receptor expression or binding with changes in circulating estrogen levels in vivo. Chronic estrogen replacement in ovariectomized rats decreases AT₁ receptor expression and binding affinity in several central sites, including the subfornical organ, a circumventricular organ (19, 20, 35, 42). In cells cultured from pituitary tumors grown with in vivo treatment of estrogen, ANG II-mediated spike elevation of the [Ca²⁺]_i response is attenuated, an indication of altered calcium mobilization mechanisms with prolonged estrogen exposure (45). Similar mechanisms could contribute to the attenuation of ANG II-mediated increases in [Ca²⁺]_i by E₂ in area postrema neurons.

E₂ modulation of ANG II-mediated increases in [Ca²⁺]_i may not be mediated by classical estrogen receptors because it was not blocked by ICI. In addition, ICI showed partial agonist effects and attenuated ANG II-evoked [Ca²⁺]_i responses. However, there is information available showing that several actions of estrogen can be independent of the classical nuclear ERs sensitive to ICI. E₂ increases nitric oxide production in human endothelial cells (44) and modulates genomic signaling pathways in murine macrophages by the activation of a membrane ER insensitive to ICI (12).

Recent reports in literature also suggest that ICI has partial agonist effects, e.g., upregulation of GAPDH mRNA in superficial endometrial cells (34), inhibition of cellular aromatase function in breast cancer cell line (MCF) cells (24), and angiogenesis (10) independent of estrogen-mediated effects. ICI has effects like that of estrogen in intact rat hearts and isolated smooth muscle, where it acts on membrane receptors to slow calcium entry into cells to dilate vessels (37). In colonic myocytes, ICI activates calcium-activated potassium currents (3). Some of the effects of estrogen have been attributed to nongenomic mechanisms. Nongenomic effects of estrogen have been defined as those which result in rapid responses within minutes of application of the hormone and are thought to require activation of membrane receptors or other cellular components to alter neuronal activity. In the present study, it is not clear if the effects of E₂ on ANG II responses involve genomic or nongenomic mechanisms or a coordination of effects mediated by both pathways. Shorter incubations of E₂ could produce similar effects on ANG II-mediated increases in intracellular calcium. The nongenomic versus genomic pathways require further clarification.

The partial agonist effect could also be dose related, because 10 µM ICI is 10 times the dose known to block the effects of E₂ on potassium currents in area postrema neurons (22). However, at the same dose, ICI was very effective in blocking E₂ effects on KCl-mediated increases in intracellular calcium and had no partial agonist effects. This further supports the notion that estrogen modulation of KCl- and ANG II-evoked increases in intracellular calcium may be mediated via different signaling pathways.

An observation in the present study is that in neurons grown in media containing dextran-stripped serum, ANG II produced significantly higher increases in [Ca²⁺]_i, which is consistent with the conclusion that estrogen alters ANG II effects. The underlying mechanisms require further investigation.

Unlike ANG II, KCl-evoked increases in intracellular calcium require depolarization of the membrane and calcium influx through voltage-gated calcium channels. Smaller KCl-evoked increases in [Ca²⁺]_i in neurons grown in estrogen-stripped media would suggest a smaller amount of calcium influx potentially due to alterations in the function of voltage-gated calcium channels or potassium channels or the number of ion channels. Several studies have shown that E₂-mediated increases in potassium currents resulted in hyperpolarization of the membrane (17, 27). Growing neurons in estrogen-stripped media could potentially reduce potassium channel conductance and ultimately change the resting membrane potentials to less negative values. The amplitude of calcium influx evoked with 60 mM KCl is potentially smaller in a neuron with a slightly depolarized membrane than a hyperpolarized membrane. The converse may be true, in neurons that showed facilitation of KCl-evoked increases in [Ca²⁺]_i when exposed to E₂ for 12 h. The type of potassium current that is being modulated by chronic E₂ exposure is not known at present. Li and Hay (22) have shown that acute administration of E₂ (50 nM) to area postrema neurons grown in normal media increases potassium currents, the majority of which was MaxiK⁺ currents. MaxiK⁺ activation is dependent on membrane potential as well as an elevation of intracellular calcium. Although it is known to modulate repetitive firing of neurons by altering afterhyperpolarization, it may not have a major influence on the resting membrane potential. However, prolonged exposure to E₂ may effect other types of potassium channels that have been previously identified in area postrema neurons (13).

It is not clear why the KCl-evoked increases in [Ca²⁺]_i in neurons incubated in E₂ plus ICI for 12 h were smaller than those of the control. It is obviously not a partial agonist effect of ICI, because ICI had no effect when applied alone. E₂, when applied alone, had a predominantly facilitatory effect. It is possible that the blockade of classical ER receptors with ICI unmasked an inhibitory effect of E₂ on the KCl-induced response. E₂ has been shown to either increase or decrease calcium currents depending on the tissue type or pathway being activated (18, 26).

In conclusion, the data from the present study provide evidence that E₂ has differential modulatory effects on ANG II- and KCl-induced increases in [Ca²⁺]_i in area postrema neurons. Differential responses to ICI reflect differences in signaling pathways. It remains to be determined if these effects are genomic or nongenomic, involving membrane ERs. Studying the interaction between estrogen and ANG II at the cellular level is important to understand how sex steroids like estrogen effect ANG II modulation of baroreflexes and other physiological functions mediated by the area postrema.

	DISCLOSURES

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This study was supported by National Heart, Lung, and Blood Institute Grant HL-62261.

	ACKNOWLEDGMENTS

 
The authors thank Lela Hall for excellent technical assistance with calcium imaging experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. Pamidimukkala, Dalton Cardiovascular Research Center, Univ. of Missouri-Columbia, 134 Research Park, Columbia, MO 65211 (E-mail: PamidimukkalaJ{at}missouri.edu).

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

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