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Endocrinology, doi:10.1210/en.2007-0073
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Endocrinology Vol. 148, No. 11 5348-5354
Copyright © 2007 by The Endocrine Society

Dual Effects of Daidzein, a Soy Isoflavone, on Catecholamine Synthesis and Secretion in Cultured Bovine Adrenal Medullary Cells

Minhui Liu, Nobuyuki Yanagihara, Yumiko Toyohira, Masato Tsutsui, Susume Ueno and Yuko Shinohara

Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan

Address all correspondence and requests for reprints to: Nobuyuki Yanagihara, Ph.D., Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yanagin{at}med.uoeh-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We recently demonstrated the occurrence and functional roles of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of daidzein, a phytoestrogen of soybeans, on catecholamine synthesis and secretion in the cells. Incubation of cells with daidzein for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine but not [14C]dihydroxyphenylalanine, in a concentration-dependent manner (10–1000 nM). The stimulatory effect of daidzein on 14C-catecholamine synthesis was not inhibited by ICI182,780, a classical estrogen receptor inhibitor. Acetylcholine, a physiological secretagogue, stimulated the synthesis of 14C-catecholamines, which was suppressed by daidzein at 1 µM. Daidzein at high concentrations (1–100 µM) suppressed catecholamine secretion induced by acetylcholine. Furthermore, daidzein (10–1000 nM) inhibited the specific binding of [3H]17β-estradiol to plasma membranes isolated from bovine adrenal medulla. The present findings suggest that daidzein at low concentrations stimulates catecholamine synthesis through plasma membrane estrogen receptors but at high concentrations inhibits catecholamine synthesis and secretion induced by acetylcholine in bovine adrenal medulla. The latter effect of daidzein may be a beneficial action on the cardiovascular system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOYBEANS HAVE BEEN traditionally consumed as food, especially in East Asian countries. Isoflavones such as daidzein and genistein are soy phytoestrogens and have estrogenic activity due to the fact that their structures are similar to the primary structure of estrogens (1). Much research attention has been paid to high dietary intake of isoflavones because of their potentially beneficial effects associated with a reduction in the risk of developing osteoporosis (2, 3), menopausal symptoms (4), high cholesterol (5), and some forms of cancer (6). Daidzein is present in high concentrations as a glycoside in many soybean products with a nonsteroidal structure (7). Several in vitro and in vivo studies have shown that daidzein has various biological actions, including a weak estrogenic or antiestrogenic effect by binding to the nuclear estrogen receptors (see reviews in Refs. 1 and 7).

Adrenal medullary cells derived from embryonic neural crests are functionally homologous to sympathetic ganglionic neurons; in the cells, our previous studies showed that Na+ influx induced by stimulation of nicotinic acetylcholine receptor-ion channels increases Ca2+ influx via voltage-dependent Ca2+ channels, a prerequisite for secretion (8) and synthesis (9) of catecholamines; in contrast, high K+ directly gates voltage-dependent Ca2+ channels to increase Ca2+ influx without increasing Na+ influx. Stimulation of catecholamine synthesis induced by acetylcholine is associated with an activation of tyrosine hydroxylase (9), which catalyzes the conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA), the rate-limiting step of catecholamine biosynthesis (10). Tyrosine hydroxylase is regulated by two different mechanisms: short-term regulation by allosteric activation (11) and long-term regulation by enzyme induction (12). In the former case, tyrosine hydroxylase is acutely regulated by various factors (11), such as enzyme phosphorylation (13).

Recently we reported that treatment of cultured bovine adrenal medullary cells with environmental estrogenic pollutants increases catecholamine synthesis probably through plasma membrane estrogen receptors (14). Furthermore, we showed the occurrence and pharmacological characterization of plasma membrane estrogen receptors in the cells and that isoflavones such as daidzein strongly inhibited the specific binding of [3H]17β-estradiol to plasma membranes, suggesting an interaction of isoflavones with the membrane estrogen receptors (15). In the present study, to determine the pharmacological actions of soy isoflavones on plasma membrane estrogen receptors, we examined the effects of daidzein on catecholamine synthesis and secretion in cultured bovine adrenal medullary cells. We found that daidzein has dual effects on catecholamine synthesis and secretion in bovine adrenal medullary cells and discussed the beneficial action of daidzein on the cardiovascular system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Oxygenated Krebs-Ringer phosphate (KRP) buffer was used throughout. Its composition is as follows (in millimoles): 154 NaCl, 5.6 KCl, 1.1 MgSO4, 2.2 CaCl2, 0.85 NaH2PO4, 2.15 Na2HPO4, and 10 glucose (adjusted to pH 7.4). Materials were obtained from the following sources: Eagle’s MEM was from Nissui Pharmaceutical (Tokyo, Japan); collagenase was from Nitta Zerachin (Osaka, Japan); calf serum, acetylcholine, and 17β-estradiol were from Nacalai Tesque (Kyoto, Japan); ERK kinase inhibitor U0126 was from Promega (Madison, WI); daidzein, H-89, and ICI 182,2780 were from Sigma-Aldrich (St. Louis, MO); L-[1-14C]tyrosine (54.45 mBq/mmol) and [2,4,6,7-3H]17β-estradiol (3515 GBq/mmol) was from PerkinElmer Life Sciences (Boston, MA); L-[U-14C]tyrosine (460 mBq/mmol), L-[3-14C]DOPA (6.8 mBq/mmol), and 45CaCl2 (0.185–1.85 GBq/mg) were from GE Healthcare U.K. Ltd. (Little Chalfont, Buckinghamshire, UK).

Isolation and primary culture of bovine adrenal medullary cells
Bovine adrenal medullary cells were isolated by collagenase digestion of adrenal medullary slices, as described previously (14). Cells were suspended in Eagle’s MEM containing 10% calf serum, 3 µM cytosine arabinoside, and several antibiotics and maintained in monolayer culture at a density of 4 x 106 cells/dish (35-mm dish; Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in 5% CO2-95% air. The cells were used for experiments between 2 and 5 d of culture.

14C-catecholamine synthesis from [14C]tyrosine or [14C]DOPA
Cells were incubated with 20 µM L-[U-14C]tyrosine (1 µCi) or L-[3-14C]DOPA (0.25 µCi) in 1.0 ml of KRP buffer in the presence or absence of various concentrations of daidzein and/or 0.3 mM acetylcholine at 37 C for 20 min (for [14C]tyrosine) or 15 min (for [14C]DOPA). After removing the incubation medium by aspiration, cells were harvested in 2.5 ml of 0.4 M perchloric acid and left standing for more than 30 min on ice to extract the radioactive catecholamines. The precipitated protein was removed by centrifugation at 1600 x g for 10 min and 14C-labeled catechol compounds were separated further by ion exchange chromatography on Duolite C-25 columns (H+-type, 0.4 x 7.0 cm) (14) and counted for radioactivity. 14C-catecholamine synthesis was expressed as the sum of the 14C-catecholamine (epinephrine, norepinephrine, and dopamine) because the ratio of [14C]epinephrine plus [14C]norepinephrine/[14C]dopamine was not significantly changed by stimulants. In control cells, the synthesis of [14C]norepinephrine plus [14C]epinephrine, [14C]dopamine, and 14C-total catecholamines are 22,000 ± 700, 20,000 ± 800, 42,000 ± 1000 dpm per 4 x 106 cells per 20 min, respectively. The ratio of [14C]norepinephrine plus [14C]epinephrine to [14C]dopamine is 1.12 ± 0.06, 1.10 ± 0.07, 1.09 ± 0.03, and 1.11 ± 0.07 in control, daidzein-treated, acetylcholine-treated, and daidzein plus acetylcholine-treated cells, respectively.

Tyrosine hydroxylase activity in situ
The cells (106 cells/well; 24-well, Falcon) were exposed to 200 µl of the KRP buffer with or without daidzein (1 µM) or ICI 182,780 (0.1 µM), supplemented with 18 µM L-[1-14C]tyrosine (0.2 µCi) for 10 min at 37 C. Upon addition of the labeled tyrosine, each well was sealed immediately with an acrylic tube capped with a rubber stopper and fitted with a small plastic cup containing 200 µl of NCS-II tissue solubilizer (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) to absorb the 14CO2 released by the cells and counted for the radioactivity (14).

Catecholamine secretion
The secretion of catecholamines was measured as described previously (16). The cells (2 x 106/dish) were washed three times with 1 ml of KRP, and they were stimulated at 37 C for 10 min with or without several concentrations of daidzein (0.1–100 µM) in the presence or absence of secretagogues. Catecholamines secreted into the medium were adsorbed to aluminum hydroxide and estimated by the ethylenediamine condensation method (17), using a fluorescence spectrophotometer (F-4010; Hitachi, Tokyo, Japan) with an excitation wavelength of 420 nm and an emission of 540 nm, respectively. Catecholamines secreted were expressed as a percentage of total catecholamines in the cell.

Measurement of 45Ca2+ influx
The influx of 45Ca2+ was measured as reported previously (8). After treatment of cells (4 x 106/dish) with daidzein for 10 min, cells were incubated with 1.5 µCi of 45Ca2+ at 37 C for 10 min in the presence or absence of acetylcholine and/or daidzein in KRP buffer. The cells were washed with ice-cold KRP buffer four times, solubilized in 10% Triton X-100, and counted for the radioactivity of 45Ca2+.

Assay of cAMP level in the cells
Cells (4 x 106/dish) were pretreated with 0.3 mM 3-isobutyl-1-methylxanthine at 37 C for 10 min and then incubated with daidzein (1 µM) or forskolin (1 µM) for another 10 min. The cells were harvested with 0.1 M HCl and centrifuged at 13,000 x g for 10 min. The cAMP level in the supernatant was measured by a cAMP RIA kit (Yamasa, Tokyo, Japan).

[3H]17β-estradiol binding to plasma membranes isolated from adrenal medulla
Plasma membranes were isolated from bovine adrenal medulla as described previously (18) with a slight modification (19). The specific binding of [3H]17β-estradiol was determined by incubating plasma membranes (30 µg of proteins) in Krebs-Ringer HEPES buffer [125 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM MgSO4, 5.6 mM glucose, 25 mM HEPES-Tris (pH 7.4)] (final volume of 200 µl) with various concentrations (1–1000 nM) of daidzein and [3H]17β-estradiol (5 nM) at 4 C for 30 min. Then [3H]17β-estradiol bound to the membranes was separated from free ligand by filtration through a GF/C glass fiber filter (Whatman, Maidstone, UK), and the filter was washed three times with the ice-cold binding buffer. Specific binding of [3H]17β-estradiol was defined as the total binding minus nonspecific binding, which was determined in the presence of 17β-estradiol (1 µM). The nonspecific binding is calculated to be 38 ± 3% of the total binding.

Statistics
Data are presented as means ± SEM. The statistical evaluation of the data were performed by ANOVA. When a significant F value was found by ANOVA, Dunnett’s or Scheffé’s test for multiple comparisons were used to identify differences among the groups. Values were considered statistically significant when P < 0.05. Statistical analyses were performed using StatView for Macintosh (version 5.0J software; Abacus Concept Inc., Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of 14C-catecholamine synthesis by daidzein in cultured bovine adrenal medullary cells
The cells were incubated with or without (1 µM) daidzein at 37 C for the indicated lengths of time (Fig. 1AGo). The basal synthesis of 14C-catecholamines (the sum of [14C]epinephrine, [14C]norepinephrine, and [14C]dopamine production) from [14C]tyrosine was linear for up to 30 min, as previously reported (20). Daidzein elicited a small (about 15–25% over the control) but significant (P < 0.05) increase in 14C-catecholamine synthesis during the incubation for 10–30 min. On the basis of this result, subsequent measurements of 14C-catecholamine synthesis were performed with 20 min of incubation. Daidzein increased 14C-catecholamine synthesis in a concentration-dependent manner, i.e. a significant increase in 14C-catecholamine synthesis was detected at 10 nM and was maximal at 100–1000 nM (Fig. 1BGo).


Figure 1
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  		FIG. 1. Time course (A) and concentration-response curve (B) of daidzein for 14C-catecholamine synthesis from [14C]tyrosine in cultured bovine adrenal medullary cells. A, Cultured cells (4 x 106/dish) were incubated with (bullet) or without ({circ}) daidzein (1 µM) at 37 C for the indicated times in 1.0 ml KRP buffer containing L-[U-14C]tyrosine (20 µM, 1 µCi). The 14C-labeled catecholamines formed are shown as the total 14C-catecholamines (epinephrine, norepinephrine, and dopamine). Data are expressed as the mean ± SEM of four experiments carried out in duplicate. B, Cells (4 x 106/dish) were incubated without ({circ}) or with various concentrations (1–1000 nM) (bullet) of daidzein at 37 C for 20 min in 1.0 ml KRP buffer containing L-[U-14C]tyrosine (20 µM, 1 µCi). The 14C-catecholamines formed were measured. Data are expressed as means ± SEM of three separate experiments carried out in triplicate. *, P < 0.05 and **, P < 0.01, compared with control.

 
Effect of daidzein and/or acetylcholine on 14C-catecholamine synthesis from [14C]tyrosine or [14C]DOPA
As shown in Fig. 2AGo, daidzein (1 µM) and acetylcholine (0.3 mM) increased the synthesis of 14C-catecholamines from [14C]tyrosine by 31 and 245% over the control, respectively. Concurrent treatment of cells with daidzein and acetylcholine did not enhance but inhibited the stimulatory effect of acetylcholine on 14C-catecholamine synthesis (84% of acetylcholine alone). To determine which step of catecholamine synthesis was stimulated by daidzein, [14C]DOPA was used as a substrate instead of [14C]tyrosine. The basal synthesis of 14C-catecholamines from [14C]DOPA was much greater than that from [14C]tyrosine (Fig. 2BGo). Neither daidzein nor acetylcholine increased the synthesis of 14C-catecholamines from [14C]DOPA, indicating that the stimulation of 14C-catecholamine synthesis caused by daidzein and acetylcholine occurs predominantly upstream of the DOPA decarboxylase step.


Figure 2
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  		FIG. 2. Effects of daidzein and/or acetylcholine on 14C-catecholamine synthesis from [14C]tyrosine (A) or [14C]DOPA (B). The cells (4 x 106/dish) were incubated with or without daidzein (1 µM) and acetylcholine (ACh) (0.3 mM) at 37 C for 20 or 15 min in the presence of L-[U-14C]tyrosine (20 µM, 1 µCi) (A) or [14C]DOPA (20 µM, 0.25 µCi) (B), respectively. The 14C-catecholamines formed were measured. Data are expressed as the mean ± SEM of four experiments carried out in triplicate. *, P < 0.05 and **, P < 0.01, compared with control; ***, P < 0.05, compared with acetylcholine alone.

 
Effects of an inhibitor of classical estrogen receptors and various inhibitors and an activator of protein kinases on 14C-catecholamine synthesis and tyrosine hydroxylase activity
To resolve whether daidzein exerts its effect on 14C-catecholamine synthesis through nuclear estrogen receptors, we used ICI182,780, a pure antagonist of classical estrogen receptors (21). ICI182,780 (100 nM), by itself, significantly increased 14C-catecholamine synthesis by 43% over the control, whereas the stimulatory effect of daidzein was not inhibited but rather enhanced by ICI182,780 (95% over the control) (Fig. 3Go). To investigate the intracellular signal of daidzein, we used forskolin as an activator of adenylate cyclase and H-89 and U0126 as inhibitors of cAMP-dependent protein kinase (protein kinase A) and ERK1/2 kinase, respectively. U0126 (1 µM) diminished the stimulatory effect of daidzein on 14C-catecholamine synthesis. Forskolin at a maximal concentration of 10 µM caused an increase in 14C-catecholamine synthesis by 309% over the control. Daidzein (1 µM) did not enhance forskolin-induced synthesis of 14C-catecholamines, suggesting a mechanism common to that of forskolin. H-89 at 10 µM, by itself, suppressed the basal synthesis and abolished the stimulatory effect of daidzein on 14C-catecholamine synthesis (Fig. 3Go). We further examined the effect of daidzein and forskolin on cAMP level in cultured bovine adrenal medullary cells. Incubation of the cells with daidzein (1 µM) resulted in a small but significant increase in cAMP level by 14 ± 2% (P < 0.05, n = 4) over the control, whereas forskolin (1 µM) produced 493 ± 6% (P < 0.001, n = 4) increase in cAMP level over the control (our unpublished observation).


Figure 3
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  		FIG. 3. Effects of an inhibitor of classical estrogen receptors and various inhibitors and an activator of protein kinases on 14C-catecholamine synthesis. The cells (4 x 106/dish) were incubated with or without daidzein (1 µM) and/or ICI182,780 (100 nM), U0126 (1 µM), forskolin (10 µM), and H-89 (10 µM) at 37 C for 20 min in the presence of L-[U-14C]tyrosine. Data are expressed as the means ± SEM of four experiments carried out in triplicate. *, P < 0.05 and ***, P < 0.01, compared with control; **, P < 0.05, compared with daidzein alone.

 
We then examined the effect of treatment with daidzein on tyrosine hydroxylase activity in cells. Incubation with daidzein (1 µM) and ICI182,780 (100 nM) for 10 min caused significant increases in tyrosine hydroxylase activity of 27 and 40%, respectively, over the activity in the control (Fig. 4Go). Incubation of cells with ICI182,780 did not abolish but rather enhanced tyrosine hydroxylase activity induced by daidzein (65% over the control).


Figure 4
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  		FIG. 4. Effects of daidzein and/or ICI182,780 on tyrosine hydroxylase activity in the cells. Cells (106/well) were preincubated in 250 µl of KRP buffer with or without daidzein (1 µM) and/or ICI182,780 (ICI) (100 nM) for 10 min and then incubated for another 10 min in the presence of L-[1-14C]tyrosine (18 µM, 0.2 µCi), and tyrosine hydroxylase activity was measured. Data are the mean ± SEM of four separate experiments carried out in triplicate. *, P < 0.05 and **, P < 0.01, compared with control; ***, P < 0.05, compared with daidzein.

 
Effects of daidzein on catecholamine secretion induced by acetylcholine or high K+
We examined the effect of daidzein on catecholamine secretion induced by secretagogues. Bovine adrenal medullary cells secreted catecholamines after the stimulation of nicotinic acetylcholine receptor-ion channels with acetylcholine, a physiological secretagogue, and voltage-dependent Ca2+ channels with 56 mM K+ (high K+) (Fig. 5Go). The catecholamines secreted after treatment with these respective stimuli corresponded to 13.5 ± 0.12 and 10.8 ± 0.09% of the total catecholamine content in the cells. Pretreatment of cells with daidzein at 1, 10, and 100 µM for 10 min significantly reduced acetylcholine-induced secretion of catecholamines to 94, 88, and 64% of acetylcholine alone (Fig. 5AGo), respectively, whereas daidzein only at 100 µM inhibited 56 mM K+-induced secretion to 85% of the level secreted after stimulation with 56 mM K+ alone (Fig. 5BGo). Acetylcholine (0.3 mM) caused an influx of 45Ca2+ (5.6 ± 0.3 nmol per 4 x 106 cells per 10 min), which was reduced by daidzein at 10 and 100 µM to 92 and 88% (P < 0.05, n = 4) of that of acetylcholine alone (our unpublished observation).


Figure 5
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  		FIG. 5. Effects of daidzein on catecholamine secretion induced by acetylcholine (A) or high K+ (B) in bovine adrenal medullary cells. Cells (2 x 106/dish) were incubated with acetylcholine (0.3 mM) (A) or high K+ (56 mM K+) (B) in the presence of various concentrations of daidzein for 10 min at 37 C. Catecholamines secreted into the medium were measured (see Materials and Methods) and expressed as percentage of total catecholamines. Data are means ± SEM of four experiments carried out in duplicate. *, P < 0.05 and **, P < 0.01, compared with control (0 µM daidzein).

 
Effect of daidzein on [3H]17β-estradiol binding to plasma membranes
Recently we reported the occurrence and characterization of [3H]17β-estradiol-specific binding to the plasma membranes isolated from bovine adrenal medulla (15). When plasma membranes were incubated with various concentrations of daidzein and [3H]17β-estradiol (5 nM), the specific binding of [3H]17β-estradiol was inhibited by daidzein in a concentration-dependent manner (10–1000 nM) (Fig. 6Go). When the result of Fig. 6Go was examined by a modified Scatchard analysis (22), it showed two components with IC50 values of 1.9 and 34.5 nM (data not presented).


Figure 6
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  		FIG. 6. Concentration-inhibition curve of daidzein for specific binding of [3H]17β-estradiol. Plasma membranes isolated form bovine adrenal medulla were incubated at 4 C for 30 min with various concentrations of daidzein in the presence of [3H]17β-estradiol (5 nM). Nonspecific binding was determined in the presence 1 µM of 17β-estradiol, and specific binding was obtained by subtracting nonspecific binding from total binding. Values shown are means ± SEM of four separate experiments carried out in triplicate. *, P < 0.05 and **, P < 0.01, compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that daidzein stimulated the basal synthesis of 14C-catecholamines from [14C]tyrosine in cultured bovine adrenal medullary cells. When [14C]DOPA was used as a substrate instead of [14C]tyrosine, daidzein failed to stimulate the synthesis of 14C-catecholamines, suggesting that the stimulation of 14C-catecholamine synthesis induced by daidzein occurs predominantly before the DOPA decarboxylase step and probably at the tyrosine hydroxylase step. Indeed, daidzein increased tyrosine hydroxylase activity in the cells. Based on these findings, it is likely that daidzein activates tyrosine hydroxylase and subsequently stimulates catecholamine synthesis. To the best of our knowledge, this is the first direct evidence showing the stimulatory effects of daidzein, a soy isoflavone, on catecholamine synthesis.

The serum concentrations of daidzein have been reported to be 72 nM in Japanese middle-aged women (23) and around 200–350 nM in Japanese people older than 40 yr (24). Furthermore, the serum levels of daidzein in humans consuming three meals per day containing soy milk or a single soy meal can reach maxima of 4.6 and 4.1 µM, respectively (25, 26). Therefore, it is likely that the concentrations used in the present study are relevant in people’s daily lives because these concentrations partially overlap with those in the plasma of individuals who consume soy products.

Estrogens exert multiple biological effects on a diverse array of target tissues. Many estrogenic actions that require hours to days to accomplish are mediated through the nuclear estrogen receptors in a genomic manner (27). In addition to this established mechanism of actions, there is growing evidence that estrogens have nongenomic actions via the activation of estrogen receptors in the plasma membrane (see reviews in Refs. 28 and 29). These actions are marked by their rapid time course and are not blocked by inhibitors of classical estrogen receptors, even though there are many reports of antiestrogen blocking agents with nongenomic responses (21, 30, 31). In the present study, the stimulatory effects of daidzein on both catecholamine synthesis and tyrosine hydroxylase activity were rapidly observed within 10 min. ICI182,780, an inhibitor of classical estrogen receptors, did not inhibit catecholamine synthesis and tyrosine hydroxylase induced by daidzein. Therefore, it is likely that daidzein activates tyrosine hydroxylase and stimulates catecholamine synthesis through a nuclear estrogen receptor-independent pathway. In our unpublished observation, daidzein and 17β-estradiol at each maximally effective concentration of 1 µM and 100 nM increased 14C-catecholamine synthesis by 26 and 48% over the control, respectively, whereas daidzein (1 µM) plus 17β-estradiol (100 nM) increased it by 42% over the control, indicating no additive effect. Therefore, it seems that daidzein and 17β-estradiol may act on the same site to stimulate catecholamine synthesis. Furthermore, our previous study (15) reported that ICI182,780 did not inhibit but rather enhanced the specific binding of [3H]17β-estradiol to plasma membranes isolated from bovine adrenal medulla, whereas daidzein strongly inhibited it, suggesting that ICI182,780 and daidzein are acting at different sites of the membrane estrogen receptors or different membrane receptors. Further study, however, is required to prove the fundamental issue how daidzein acts on the plasma membrane estrogen receptors or other membrane receptors.

Estrogens have been shown to rapidly trigger a variety of second-messenger signaling events including the stimulation of cAMP (32) and calcium mobilization (33), the generation of inositol phosphate (34), and the activation of the ERK1/2 (35). On the other hand, previous reports have shown that tyrosine hydroxylase can be phosphorylated and activated by several multiple protein kinases (11) such as protein kinase A in bovine adrenal medullary cells (36) and ERK1/2 in rat pheochromocytoma PC12 cells (37) or bovine adrenal medullary cells (38). We also reported that 17β-estradiol stimulates catecholamine synthesis via the activation of ERK1/2 in cultured bovine adrenal medullary cells (15). In the present study, U0126 (10 µM), an inhibitor of ERK1/2 activation, diminished the stimulatory effect of daidzein on the catecholamine synthesis. Furthermore, daidzein at maximal concentration did not enhance forskolin-induced synthesis of 14C-catecholamine. H-89, an inhibitor of protein kinase A, abolished the stimulatory effect of daidzein. We also observed a small but significant increase in cAMP level induced by daidzein. Therefore, daidzein may stimulate catecholamine synthesis through protein kinase A- and/or ERK1/2-dependent pathways.

Recently several types of estrogen receptors in plasma membranes have been reported, including classical nuclear estrogen receptors (39), a novel member of the estrogen receptor family ER-X (40), and GPR30 that has a high homology to the G protein-coupled receptor superfamily in breast cancers (41). Thomas et al. (42) reported that [3H]17β-estradiol binding to GPR30 in breast cancer cells was inhibited by ICI182,780 and tamoxifen but by neither 17{alpha}-estradiol nor other steroid hormones such as corticosterone and testosterone at the concentration of 1 µM. In our previous study (15), we pharmacologically characterized the specific binding sites of [3H]17β-estradiol to plasma membranes isolated from bovine adrenal medulla and found that tamoxifen and ICI182,780 had little effect or stimulatory effect on [3H]17β-estradiol binding, whereas the binding of [3H]17β-estradiol was inhibited by 17{alpha}-estradiol, corticosterone, and testosterone at 0.1–10 µM, which were 100-fold higher concentrations than that of 17β-estradiol. On the other hand, ER-X was activated equally well by picomolar concentrations of 17{alpha}- and 17β-estradiol to increase the phosphorylation of ERK1/2 in mouse neocortical cells. Therefore, we considered that the pharmacological properties of the plasma membrane estrogen receptors in adrenal medullary cells may be different from that of several types of plasma membrane estrogen receptors reported previously. However, we must keep in mind that such comparisons by using [3H]17β-estradiol binding may not be valid for other tissues or the classical estrogen receptors in different structural environments. To know how daidzein actually interacts with the membrane estrogen receptors, further studies to identify the plasma membrane estrogen receptors are required in feature.

The high-affinity binding sites of [3H]17β-estradiol (5 nM) were competed for daidzein in a concentration-dependent manner in the range of 10–1000 nM, which is similar to that observed in the stimulation of catecholamine synthesis by daidzein. On the other hand, daidzein inhibited acetylcholine-induced catecholamine secretion at concentrations ranging from 1 to 100 µM. These findings may suggest that daidzein inhibits the catecholamine secretion induced by acetylcholine through low-affinity binding sites of [3H]17β-estradiol or a receptor-independent pathway.

Recently isoflavones such as daidzein and genistein were reported to play a role in the prevention of cardiovascular diseases, reproductive cancers, and menopausal symptoms such as osteoporosis and hot flashes (43, 44, 45, 46). The cardioprotective ability of these isoflavones has been attributed partially to their ability to lower cholesterol (47, 48) and cardiovascular disease risk (49). In the present study, daidzein (1 µM) increased 14C-catecholamine synthesis by 20–30% over the control, whereas the same concentration (1 µM) of daidzein inhibited catecholamine synthesis and secretion induced by acetylcholine. The present findings suggest that daidzein at high concentrations (≥1 µM) attenuates the catecholamine synthesis and secretion induced by stress or emotional excitation that induces the stimulation of splanchnic nerves and subsequently the adrenal medulla. Catecholamines play a pivotal role in the regulation of normal functions in cardiovascular systems, whereas stress-induced over expression of catecholamines would contribute to the involvement and augmentation of cardiovascular diseases such as heart failure, atherosclerosis, coronary heart disease, and hypertension (50). Indeed, chronic heart failures are associated with activation of the sympathetic nervous system as manifested by increased circulating level of norepinephrine and increased regional activity of the sympathetic nervous system (50, 51, 52). Thus, our findings may partially explain the cardiovascular protective effects of daidzein. Nevertheless, the present findings add to our understanding of soy food functions by describing new pharmacological actions of an isoflavone, daidzein. To confirm the effects of daidzein on catecholamine synthesis and secretion, further in vivo studies of the administration of daidzein to animals or humans are required.

In summary, we have demonstrated that daidzein at a range of human serum concentrations stimulates catecholamine synthesis through plasma membrane estrogen receptors but at high concentrations inhibits catecholamine synthesis and secretion induced by acetylcholine in the bovine adrenal medulla and probably in the sympathetic neurons.


   Acknowledgments
 
We thank Ms. Kazumi Tanaka for her expert technical assistance.


   Footnotes
 
This work was supported in part by Grants-in-Aid (17590111 and 18602004) for Scientific Research (C) from the Japan Society for the Promotion of Science and a grant from the Smoking Research Foundation.

Disclosure Statement: All authors have nothing to disclose.

First Published Online August 23, 2007

Abbreviations: DOPA, Dihydroxyphenylalanine; KRP, Krebs-Ringer phosphate; protein kinase A, cAMP-dependent protein kinase.

Received January 19, 2007.

Accepted for publication August 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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