Natural estrogens have a wide array of biological actions not only on the female reproductive system but also on metabolic homeostasis, cell proliferation and differentiation. The long-term genomic effects of estrogens are known to be mediated through classical nuclear receptors such as estrogen receptor-α (ER-α) and -β (ER-β) (Green et al., 1986; Krust et al., 1986; Mosselman et al., 1996). In addition to this established mechanism of action, much evidence has been accumulated that estrogens also have non-genomic actions via the activation of plasma membrane estrogen receptors (Wehling 1997; Falkenstein et al., 2000). Indeed, the cell membrane estrogen receptors have been extensively studied, resulting in the identification of several types of membrane estrogen receptors, such as ER-α, its S-palmitoylated ER variant (Li et al., 2003; Wyckoff et al., 2001), ER-X (Toran-Allerand et al., 2002; Qui et al., 2006), and GPR30 (Thomas et al., 2005; Revankar et al., 2005). Among these, GPR30 may be the most plausible candidate for a membrane estrogen receptor that regulates the various functions induced by estrogens (Funakoshi et al., 2006; Filardo et al., 2007), although the precise cellular localization and functions of GPR30 remain controversial (for review see, Mizukami 2010).
Soybeans have traditionally been consumed as food, especially in East Asian countries. Isoflavones such as daidzein and genistein are soy phytoestrogens and have a weak estrogenic activity due to the fact that their structures are similar to the primary structure of estrogens (Kurzer and Xu, 1997). Recent research attention has been paid to the high dietary intake of isoflavones because of their potentially beneficial effects associated with a reduction in the risk of developing cardiovascular diseases (Arjmandi et al., 1997), osteoporosis (Adlercreutz et al., 1993; Toda et al., 1999), menopausal symptoms (Adlercreutz et al., 1992), and some forms of cancers (Messina et al., 1994). These effects are considered to be mediated by binding to the nuclear estrogen receptors (Kurzer and Xu, 1997; Murkies et al., 1998).
Adrenal medullary cells are derived from the embryonic neural crest and share many properties with sympathetic postganglionic neurons. In cultured bovine adrenal medullary cells, our previous studies have shown that carbachol, a derivative of acetylcholine, induced 22Na+ influx via voltage-dependent Na+ channels, and then increased 45Ca2+ influx via voltage-dependent Ca2+ channels, a prerequisite for secretion (Wada et al., 1985; Yanagihara et al., 1996) and synthesis (Yanagihara et al., 1987) of catecholamines.Since the mechanism of stimulation of catecholamine synthesis and secretion in adrenal medullary cells are thought to be similar to those of noradrenaline in the sympathetic neurons, adrenal medullary cells have provided a good model for detailed analysis of the actions of cardiovascular drugs, such as α2-adrenergic agonists (Yanagihara et al., 1987), natriuretic peptides (Yanagihara et al., 1991), carvedilol (Kajiwara et al., 2002) and pimobendan (Toyohira et al., 2005).
In our previous studies, treatment of bovine adrenal medullary cells with environmental estrogenic pollutants such as p-nonylphenol and bisphenol A stimulated catecholamine synthesis and tyrosine hydroxylase activity, probably through plasma membrane estrogen receptors (Yanagihara et al., 2005). Indeed, we demonstrated the occurrence and functional roles of unique estrogen receptors in the plasma membranes isolated from bovine adrenal medullary cells (Yanagihara et al., 2006). 17β-Estradiol stimulated catecholamine synthesis via activation of extracellular signal-regulated kinases (ERKs) through the plasma membrane estrogen receptors.
The present review summarizes the current knowledge of pharmacological effects of daidzein and genistein on catecholamine signaling, such as catecholamine synthesis and secretion in cultured bovine adrenal medullary cells and noradrenaline reuptake by SK-N-SH cells and by COS-7 cells transiently transfected with noradrenaline transporter.
2. Dual effects of daidzein on catecholamine synthesis and secretion
Incubation of bovine adrenal medullary cells with daidzein for 20 min resulted in a small increase in 14C-catecholamine synthesis from [14C]tyrosine (Fig. 1A). The stimulatory effect of daidzein on 14C-catecholamine synthesis was observed to be concentration-dependent (10-1000 nM). Daidzein inhibited the specific binding of [3H]17β-estradiol in a concentration (10-1000 nM)-dependent manner (Fig. 1B) similar to that of 14C-catecholamine synthesis. From these results, it is likely that daidzein stimulates catecholamine synthesis via activation of membrane estrogen receptors in bovine adrenal medullary cells. Previous studies have shown that the serum concentrations of daidzein are around 200-350 nM in Japanese people older than 40 years (Morton et al., 2002) and that the serum levels of daidzein in humans consuming three meals per day, including one meal containing soybeans, can reach a maximum of 4.1 μM (King and Bursill, 1998). It seems that the concentrations of daidzein 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.
Daidzein (1 μM) and acetylcholine (0.3 mM) increased 14C-catecholamine synthesis form [14C]tyrosine by 31% and 245% over the control levels, respectively (Fig. 2A). Concurrent treatment of cells with daidzein and acetylcholine did not enhance but significantly inhibited the stimulatory effect of acetylcholine on 14C-catecholamine synthesis (84% of acetylcholine). To determine which step of catecholamine synthesis was enhanced by daidzein, [14C]DOPA was used as a substrate instead of[14C]tyrosine (Fig. 2B). Neither daidzein nor acetylcholine increased 14C-catecholamine synthesis from [14C]DOPA, suggesting that the increase in catecholamine synthesis induced by daidzein and acetylcholine occurs predominantly upstream of the DOPA decarboxylase step, i.e., tyrosine hydroxylase step. Indeed, incubation of cells with daidzein (1 μM) for 10 min resulted in a significant increase in tyrosine hydroxylase activity of 27% over the control (Fig. 3B). ICI182,780 (100 nM), an inhibitor of nuclear estrogen receptors when used alone, increased 14C-catecholamine synthesis as well as tyrosine hydroxylase activity, and did not abolish but rather enhanced both stimulatory effects of daidzein (Fig. 3A and B). Furthermore, ICI182,780 enhanced the specific binding of [3H]17β-estradiol to plasma membranes isolated from bovine adrenal medulla (data not presented). This gives rise to the possibility that daidzeinand ICI182,780 act synergistically on different sitesof the membrane estrogen receptors.
We next studied the effect of daidzein on catecholamine secretion induced by acetylcholine. The catecholamine secretion induced by acetylcholine corresponded to 13.5% of the total catecholamine content in the cells. Daidzein (1, 10, and 100 μM) significantly suppressed catecholamine secretion inducedby acetylcholine to 94%, 88%, and 64% of that by acetylcholine alone (Fig. 4), respectively. These findings suggest that daidzein has dual effects on catecholamine synthesis and secretion, i.e., at low concentrations (10-100 nM) daidzein activates tyrosine hydroxylase activity and stimulates catecholamine synthesis, but at high concentrations (≧1 μM) it attenuates catecholamine synthesis and secretion induced by acetylcholine.
3. Up-regulation of noradrenaline transporter function by genistein
Treatment of SK-N-SH cells (a human neuroblastoma cell line) with genistein, another soy isoflavone, for 20 min stimulated [3H]noradrenaline uptake by the cells in a bell-shaped concentration-dependent manner (0.1-10 μM), whereas neither daidzein nor cumestrol, another phytoestrogen, did so (Fig. 5A). Genistein (0.01-10 μM) also stimulated [3H]noradrenaline uptake by COS-7 cells transiently transfected with noradrenaline transporter (Fig.5B). In Japanese middle-aged women, the dietary intake of genistein was reported to be 111.6 μmol/day/capita (30.1 mg/day/capita), and the median plasma concentration of genistein was 206 nM (Arai et al., 2000). Since Asian individuals generally consume more soy foods than do people in developed Western countries, the mean concentrations of genistein in Japanese and UK men were 493 nM and 33 nM, respectively (Morton et al., 2002). Furthermore, a previous paper (Adlercreutz et al., 1993) reported that the plasma concentration of genistein exceeded 2400 nM in one Japanese man. Therefore, it seems that the genistein concentrations used in the present study are nutritionally (or pharmacologically) relevant.
From Eadie-Hofstee analysis of [3H] noradrenaline uptake, genistein (10 μM) caused a significant increase in the maximal velocity (Vmax) of noradrenaline transport with little change in the Michealis-Menten constant (Km) value (Fig. 6A). Scatchard analysis of [3H]nisoxetine binding to COS-7 cells transiently transfected with noradrenaline transporter showed that genistein increases the maximal binding (Bmax) without any change in the dissociation constant (Kd) (Fig. 6B).
To test the involvement of nuclear estrogen receptors (ER-α and ER-β) in the stimulatory effect of genistein on noradrenaline transport, we used ICI182,780, an inhibitor of both ER-α and ER-β. COS-7 cells transfected with noradrenaline transporters were preincubated with ICI182,780 (100 nM), and then incubated for another 10 min with [3H]noradrenaline in the presence or absence of genistein (100 nM) or 17β-estradiol (100 nM). ICI182,780 by itself stimulated [3H]noradrenaline uptake by the cells (data not presented). ICI182,780 did not suppress but rather enhancedgenistein-induced [3H]noradrenaline uptake. Sodium orthovanadate (50 μM), an inhibitor of protein tyrosine phosphatase, significantly inhibited [3H]noradrenaline uptake (data not shown). Although genistein is known to be an inhibitor of tyrosine kinases, daidzein, an inactive analogue of genistein against tyrosine kinase, had little effect on [3H]noradrenaline uptake by SK-N-SH cells (Fig. 5A), suggesting an involvement of tyrosinekinase, but not of membrane estrogen receptors. Furthermore, the stimulatory effects on [3H]noradrenaline uptake were observed by treatment with tyrophostin 25, an inhibitor of epidermal growth factor receptor tyrosine kinase (Fig. 7A), whereas PP2, an inhibitor of the soluble-type src-family of tyrosine kinases, did not affect it (Fig. 7B). From these findings, it is suggested that genistein increases the activity of noradrenaline transporter, probably through processes involving receptor-type protein tyrosine phosphorylation.
4. Pharmacological significance of soy isoflavone’s effects on catecholamine system
Soy isoflavones (daidzein and genistein) are present at high concentrations as a glycoside in many soybeans and soy foods such as miso, tofu, and soy milk. Several lines of accumulating evidence have indicated that soy isoflavones play a role in the prevention of cardiovascular diseases, reproductive cancers, and menopausal symptoms (Potter et al., 1998; Cassidy et al., 1999; Upmalis et al., 2000; Ishimi et al., 1999). The cardioprotective ability of these isoflavones has been attributed partially to their ability to lower cholesterol (Wong et al., 1998; Yamakoshi et al., 2000) and cardiovascular disease risk (Lichtenstein 1998). In the present review, daidzein (0.01-1.0 μM) stimulated catecholamine synthesis by 20~30% over the control, suggesting that daidzein at nutritionally relevant concentrations strengthens the catecholamine system in the adrenal medulla and probably in the sympathetic neurons.
On the other hand, daidzein at high concentrations (over 1.0 μM) suppresses catecholamine synthesis and secretion induced by the physiological secretagogue acetylcholine, suggesting that daidzein attenuates the catecholamine synthesis and secretion induced by stress and emotional excitation, thus causing the stimulation of sympathetic nerves and the adrenal medulla. Genistein up-regulates the noradrenaline transporter function. This suggests that genistein also stimulates the termination of neurotransmission by the reuptake of noradrenaline released into the extracellular milieu and suppresses the sympathetic nerve activity. Although catecholamines play a pivotal role in the regulation of normal functions in cardiovascular systems, prolonged stress-induced over-expression of endogenous catecholamines may contribute to the involvement and augmentation of cardiovascular diseases such as heart failure, atherosclerosis, coronary heart disease, and hypertension. Indeed, chronic heart failure is associated with activation of the sympathetic nervous system as manifested by increased circulating catecholamines and increased regional activity of the sympathetic nervous system (Freedman and Lefkowitz, 2004; Westfall and Westfall 2005). Furthermore, it has been shown that up-regulation of adrenal medullary G protein-coupled receptor kinase 2 is a very important mechanism for mediating the sympathetic hyperactivity and circulating catecholamine levels that accompany and aggravate chronic heart failure (Lymperopoulos et al., 2007). The present findings on the effects of daidzein and genistein in catecholamine system may partially explain the cardiovascular protective effects of soy isoflavones.
5. Future perspectives
What are the major pending problems or questions revealed by the present study? While the in vitro effects of soy isoflavone have been well clarified using cultured bovine adrenal medullary cells, SK-N-SH cells or COS-7 cells transiently transfected with noradrenaline transporter, the in vivo effects are not as clear. Therefore, to confirm the effects of soy isoflavone on catecholamine synthesis, secretion and reuptake, further in vivo studies on the effects of administration of daidzein and genistein to animals or humans will be needed in the near future.
Finally, a question arises as to how best to demonstrate the protective effects of soy isoflavone on stress-induced catecholamine synthesis and secretoin? The anti-depressive or anxiolytic effectsof soy isoflavone should be examined using laboratory animals under various stress conditions. Analysis with in vivo studies will provide more conclusive information and add new pharmacological actions of soy isoflavone on catecholamine signaling.
6. Concluding remarks
Daidzein and genistein are major natural phytoestrogens found in soybeans. In the present review, we have demonstrated that daidzein stimulates catecholamine synthesis at low concentrations similar to those at which daidzein inhibits the specific binding of [3H]17β-estradiol to the membrane receptors, suggesting that daidzein at low concentrations enhances catecholamine synthesis probably through plasma membrane estrogen receptors (Liu et al., 2007). However, daidzein at high concentrations (1-100 μM) inhibited catecholamine synthesis and secretion induced by acetylcholine, the physiological secretagogue. The latter findings suggest that daidzein at high concentrations suppresses the catecholamine synthesis and secretion induced by stress or emotional excitation that induces the stimulation of the splanchnic nerves and subsequently the adrenal medulla. In addition to daidzein’s effects, genistein also increases the activity of the noradrenaline transporter, suggesting an enhancement of termination of noradrenaline transmission at the sympathetic nerve terminals. Although endogenous catecholamines play an important role in the regulation of normal functions in the cardiovascular system, stress-induced over expression of catecholamines may contribute to the involvement and augmentation of cardiovascular disorders The present findings would support the idea that soy isoflavone suppress excessive stress-induced hyperactivity of the sympatho-adrenal system and thereby protect the cardiovascular system (Yanagihara et al., 2008).
This work was supported in part by a grant from Grant-in-Aids (11839030, 20611020, and 20590129) for Scientific Research (C) from the Japan Society for the Promotion of Science, and a grant from the University of Occupational, Environmental Health for Advanced Research and the Smoking Research Foundation.
8. Conflict of interest
The authors have declared no conflict of interest.