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CARDIOVASCULAR

Soy Isoflavones Improve Endothelial Function in Spontaneously Hypertensive Rats in an Estrogen-Independent Manner: Role of Nitric-Oxide Synthase, Superoxide, and Cyclooxygenase Metabolites

Department of Pharmacology, School of Pharmacy, University of Granada, Granada, Spain (R.V., M.G., I.C.V., M.S., A.Z., J.D.); and Department of Pharmacology, School of Medicine, University Complutense, Madrid, Spain (F.P.-V.)

Received March 1, 2005; accepted June 10, 2005.

	Abstract

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The aim of this study was to analyze the effects of the isoflavones genistein and daidzein, and the mammalian estrogen 17-estradiol on endothelial function in isolated aortic rings from male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Relaxation to acetylcholine on precontracted rings was impaired and endothelium-dependent contraction to acetylcholine in aortic rings was increased in SHR compared with WKY. Aortic NADPH-stimulated release and prostaglandin (PG)H₂ production evoked by acetylcholine were increased, whereas nitric-oxide synthase activity was reduced in SHR versus WKY. Genistein, daidzein, or 17-estradiol enhanced the relaxant response to acetylcholine and decreased the endothelium-dependent vasoconstrictor responses to acetylcholine in SHR, but not in WKY, and these effects were not modified by the estrogen receptor antagonist ICI 182,780 (7,17-[9[(4,4,5,5,5-pentafluoropentyl)-sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol). Moreover, isoflavones enhanced nitric-oxide (NO) synthase activity and inhibited NADPH-stimulated roduction and endothelial release of PGH₂. The contractions induced by the TP receptor agonist U46619 [GenBank] (9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂) in denuded aortic rings were inhibited by genistein, daidzein, and 17-estradiol in both strains. In conclusion, the isoflavones genistein and daidzein and 17-estradiol restore endothelial function in male SHR through estrogen receptor-independent mechanisms. Increased NO production and protection of NO from -driven inactivation might be involved in the improvement of vascular relaxation to acetylcholine in aortic rings from SHR. Moreover, isoflavones and 17-estradiol inhibited aortic endothelium-dependent contraction to acetylcholine in SHR by reducing the endothelial PGH₂ release and its vasoconstrictor response.

Phytoestrogens are ubiquitous, nonsteroidal, plant-derived compounds exhibiting both estrogen agonist and antagonist activities (Kurzer and Xu, 1997). Soybean is a rich source of the isoflavone phytoestrogens genistein (4',5,7-trihydroxyisoflavone) and daidzein (4',7-dihydroxyisoflavone). Soy-enriched diets are associated with low cardiovascular risk, which has been attributed to their effects on plasma lipoproteins, as well as direct effects on the vessel wall (Anderson et al., 1999). Genistein and daidzein are ligands for estrogen receptor and , but they both show greater affinity for the estrogen receptor (Makela et al., 1999). Besides its activity on estrogen receptors, the effects of genistein have been attributed as well to its inhibitory properties on a broad range of tyrosine kinases (Ogawara et al., 1989).

In vitro, genistein enhances the dilator response to acetylcholine in coronary arteries from atherosclerotic female macaques (Honore et al., 1997) and in aortae from ovariectomized rats (Squadrito et al., 2000). In vivo, genistein has also been reported to improve endothelial function in several animal models (Bermejo et al., 2003), including SHR (Kitayama et al., 2002). Because genistein or soy supplements have been proven to be effective in protecting endothelial dysfunction in men and women, in some but not all studies (Simons et al., 2000; Squadrito et al., 2002), a better understanding of the mechanisms of action would be needed so that products or therapeutic interventions might be improved to treat hypertension in both males and females in the future.

We hypothesized that genistein and daidzein may influence in vitro the endothelial dysfunction characteristic of SHR. Thus, we analyzed their effects on endothelial function and the potential mechanisms involved: NO synthase activity, production of ROS, and vasoconstrictor prostanoid synthesis and activity. The role of estrogenic mechanisms was studied by comparing their effects with those of 17-estradiol and by using a specific estrogen receptor antagonist. We selected male animals to analyze the female sex-independent effects of phytoestrogens, at concentrations in the range of those found in plasma after consumption of a soy-rich diet (Manach et al., 2004).

	Materials and Methods

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All the experiments were performed in accordance with Institutional Guidelines for the ethical care of animals (Animals Acts, Scientific Procedures, 1986). Twenty-four-week-old male SHR and WKY (300-350 g) were used. Rats were injected with a lethal dose of pentobarbitone sodium (120 mg kg^-1 intraperitoneal) and exsanguinated.

Isolated Vessel Contractility. Descending thoracic aortic rings with or without endothelium were mounted in Krebs' solution for isometric tension recording as described previously (Duarte et al., 2001). The relaxant effects of acetylcholine were analyzed in arteries treated for 20 min with genistein, daidzein, 17-estradiol (10 µM), or DMSO (vehicle) in the absence or presence of the estrogen receptor antagonist ICI 182,780, at its known inhibitory concentration of 1 µM (Wakeling et al., 1991), or superoxide dismutase (100 U/ml), and then contracted by phenylephrine (1 µM), and finally a cumulative concentration-response curve to acetylcholine was constructed (Duarte et al., 2001). In other aortic rings, the effects of genistein, daidzein, 17-estradiol (10 µM), or DMSO (vehicle) on the endothelium-independent relaxant responses induced by sodium nitroprusside were analyzed in endothelium-denuded arteries contracted by phenylephrine (1 µM). In another set of experiments, the contractile effects of acetylcholine were analyzed in rings initially contracted by 80 mM KCl. After washing, rings were treated for 20 min with the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 100 µM) plus the isoflavones, 17-estradiol, or DMSO, and finally a cumulative concentration-response curve to acetylcholine was constructed (Duarte et al., 2001). The effects of the isoflavones, 17-estradiol, or vehicle were also studied on the contractions evoked by the TP receptor agonist U46619 [GenBank] in endothelium-denuded vessels. In preliminary experiments, both the relaxant and contractile responses to acetylcholine were fully abolished by mechanical removal of endothelium as reported previously (Iwama et al., 1992).

Prostanoid Production. PGH₂, the unstable precursor of prostaglandins and thromboxanes, isomerizes to PGE₂ under nonreducing conditions (ethanol), but it is converted to PGF₂ under reducing conditions (SnCl₂). Therefore, by measuring the difference in PGF₂ in a sample of aortic medium placed in ethanol versus that reduced with SnCl₂, it is possible to estimate the amount of PGH₂ released (Ge et al., 1995). After equilibration for 1 h in 3 ml of oxygenated Krebs' solution at 37°C, paired segments of WKY and SHR aortae were incubated in the presence of genistein, daidzein, 17-estradiol, or DMSO for 20 min and then stimulated by 100 µM acetylcholine for 5 min in the presence of L-NAME (100 µM). Thromboxane B₂ and PGF₂ were measured by enzyme immunoassay [Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK) and Assay Designs, Inc. (Ann Arbor, MI), respectively].

Vascular Nitric-Oxide Synthase Activity. NO synthase activity in aortic homogenates was determined by monitoring the conversion of L-[³H]arginine to L-[³H]citrulline (Mitchell et al., 1991). Briefly, intact aortic rings isolated from SHR and WKY were homogenized on ice, in a buffer containing 10 mM HEPES, pH 7.4, sucrose (0.32 M), EDTA (100 µM), dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (1 mg/ml), and leupeptin (10 µg/ml). Sample homogenates were incubated at room temperature for 30 min in Tris buffer (50 mM; pH 7.4) in the presence of NADPH (1 mM), tetrahydrobiopterin (10 µM), CaCl₂ (2 mM), L-valine (10 mM), calmodulin (30 nM), and a mixture of unlabeled and L-[³H]arginine (10 µM; 1 µCi/ml). Incubations were performed in the presence of genistein, daidzein, 17-estradiol, or DMSO and were concluded by addition of 1 ml of HEPES (20 mM; pH 5.5) buffer containing EGTA (1 mM) and EDTA (1 mM). L-[³H]Citrulline was separated from arginine by adding 1.5 ml of a 1:1 suspension of cation exchange resin (Dowex AG50 W-X8; Sigma Chemical, Alcobendas, Madrid, Spain) in water. The radioactivity was measured in the supernatants by liquid scintillation counting.

Aortic Superoxide Anions () Production. release in intact aortic segments was quantified by lucigenin chemiluminescence, as described previously (Ohara et al., 1993). Aortic rings from WKY or SHR were incubated for 30 min at 37°C in HEPES-containing physiological salt solution, pH 7.4, of the following composition: 119 mM NaCl, 20 mM HEPES, 4.6 mM KCl, 1 mM MgSO₄, 0.15 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 1.2 mM CaCl₂, and 5.5 mM glucose. Aortic production of was stimulated by addition of NADPH (100 µM). Incubations were performed in the presence of genistein, daidzein, 17-estradiol, diphenylene iodonium, superoxide dismutase, or DMSO. Rings were then placed in tubes containing physiological salt solution in the presence of these agents, and with or without NADPH, and lucigenin was injected automatically at a final concentration of 5 µM. Changes in release were determined by measuring luminescence over 200 s in a scintillation counter (Lumat LB 9507, Berthold, Germany) in 5-s intervals. release is expressed as relative luminescence units per minute per milligram of dry aortic tissue.

Drugs. Drugs were from Sigma Chemical, except for ICI 182,780, which was from Tocris Cookson Inc. (Bristol, UK). Stock solutions of genistein, daidzein, estradiol, and ICI 182,780 were prepared in DMSO, and all other drugs were prepared in distilled deionized water.

Analysis of Results. Results are expressed as means ± S.E.M. Statistically significant differences were calculated by Student's t test or a one-way analysis followed a Newman-Keuls post hoc test. p < 0.05 was considered statistically significant.

	Results

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Effects of Isoflavones on Acetylcholine-Induced Relaxations. In rings precontracted with phenylephrine, the relaxant response induced by acetylcholine was impaired in SHR compared with WKY (Fig. 1; p < 0.01). In aortae from WKY, genistein, daidzein, or 17-estradiol did not change the relaxation to acetylcholine (Fig. 1, A-C). However, they significantly enhanced the relaxant response to acetylcholine in rings from SHR (p < 0.05 versus vehicle; Fig. 1, D-F), restoring it to values similar to those observed in WKY rats. The pure estrogen receptor antagonist ICI 182,780 had no effect on the relaxant response induced by acetylcholine in either the absence or presence of genistein, daidzein, and 17-estradiol in SHR (Fig. 2). Acetylcholine-induced relaxation in SHR aortic rings was significantly increased by adding superoxide dismutase to the bath. However, superoxide dismutase did not alter the improvement in the relaxant response to acetylcholine induced by isoflavones or 17-estradiol (Fig. 3). In endothelium-denuded vessels from SHR, the relaxant responses to sodium nitroprusside were unaltered by previous incubation with genistein, daidzein, or 17-estradiol (Fig. 4).

View larger version (30K): [in this window] [in a new window]   Fig. 1. Effects of genistein, daidzein, and 17-estradiol, all at 10 µM, on the endothelium-dependent relaxant effects of acetylcholine in phenylephrine-contracted arteries from WKY or SHR. Values are means ± S.E.M. (n = 7-10). *, p < 0.05; **, p < 0.01 drug-treated versus vehicle (DMSO)-treated rings. The insets show the contractile tension induced by phenylephrine in the presence (solid columns) or absence (open columns) of drugs before the addition of acetylcholine.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effects of ICI 182,780 (1 µM) on genistein-, daidzein-, and 17-estradiol-enhanced (10 µM) relaxant response to acetylcholine in aortic rings from SHR. Values are means ± S.E.M. (n = 6-10). The dashed lines in A to C indicate the curve obtained with vehicle (from Fig. 1) for comparative purposes.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effects of superoxide dismutase (100 U/ml) on genistein-, daidzein-, and 17-estradiol-enhanced (10 µM) relaxant response to acetylcholine in aortic rings from SHR. Values are means ± S.E.M. (n = 5-10). The dashed lines in A to C indicate the curve obtained with vehicle (from Fig. 1) for comparative purposes.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effects of genistein, daidzein, and 17-estradiol, all at 10 µM, on the relaxant effects of sodium nitroprusside (SNP) in phenylephrine-contracted endothelium-denuded arteries from SHR. Values are means ± S.E.M. (n = 6-7). *, p < 0.05; **, p < 0.01 drug-treated versus vehicle (DMSO)-treated rings. The insets show the contractile tension induced by phenylephrine in the presence (solid columns) or absence (open columns) of drugs before the addition of nitroprusside.

Effects of Isoflavones on NO Synthase Activity. NO synthase activity measured by the conversion of L-[³H]arginine to L-[³H]citrulline was lower in aortic homogenates from SHR (38% of that in WKY; p < 0.01). Incubation with genistein, daidzein, and 17-estradiol increased NO synthase activity in both WKY and SHR (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Effects of genistein, daidzein, and 17-estradiol, all at 50 µM, on NO synthase activity in aortic homogenates from WKY and SHR measured by the conversion of L-[3H]arginine to L-[3H]citrulline. Values are means ± S.E.M. (n = 8). **, p < 0.01 versus WKY, , p < 0.05 drug-treated versus DMSO-treated rings.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effects of genistein, daidzein, and 17-estradiol, all at 50 µM, on the basal and NADPH (100 µM)-stimulated production in arteries from WKY or SHR. Values are means ± S.E.M. (n = 7-10). **, p < 0.01 versus WKY; , p < 0.05 drug-treated versus vehicle-treated rings.

Effects of Isoflavones on Vascular Production.

Effects of Isoflavones on Acetylcholine-Induced Contractions. Acetylcholine induced a contractile response in aortic rings from SHR incubated with L-NAME that was almost absent in WKY rats (Fig. 5). This response was abolished by treatment with the cyclooxygenase inhibitor indomethacin (10 µM; not shown). Genistein, daidzein, and 17-estradiol, inhibited the contraction produced by acetylcholine in SHR in a concentration-dependent manner (Fig. 7, A-C). Incubation with the pure estrogen receptor antagonist ICI 182,780 did not prevent the reduction on contractile response to acetylcholine induced by 50 µM genistein, daidzein, and 17-estradiol in SHR (Fig. 8, A-C).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Effects of genistein, daidzein, and 17-estradiol, all at 10 and 50 µM, on the endothelium-dependent contractile effects of acetylcholine in L-NAME (100 µM)-treated arteries from WKY or SHR. Values are means ± S.E.M. (n = 7-10). **, p < 0.01 versus WKY; , p < 0.05; , p < 0.01 drug-treated versus vehicle-treated rings.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effects of ICI 182,780 (0.1 µM) on genistein-, daidzein-, and 17-estradiol-inhibited (all at 50 µM) contractile response to acetylcholine in L-NAME (100 µM)-treated aortic rings from SHR. Values are means ± S.E.M. (n = 5-10). The dashed lines in A to C indicate the curve obtained with vehicle for comparative purposes.

Effects of Isoflavones on the Release of TXA₂, PGF₂, and PGH₂. The aortic release of TXA₂, PGF₂, and PGH₂ from WKY and SHR was determined under basal conditions and after stimulation with acetylcholine (Fig. 9). In WKY, acetylcholine or endothelium removal did not significantly modify the release of TXA₂, PGF₂, and PGH₂. The basal aortic production of TXA₂ in SHR was increased compared with WKY, but acetylcholine was unable to induce a further increase. Genistein, daidzein, and 17-estradiol decreased the production of TXA₂ in SHR. In aortae from SHR, the release of both PGF₂ and PGH₂ was augmented significantly upon stimulation with acetylcholine in an endothelium-dependent manner, and this increase was significantly inhibited in aortae exposed to genistein, daidzein, and 17-estradiol.

View larger version (21K): [in this window] [in a new window]   Fig. 9. Effects of genistein, daidzein, and 17-estradiol, all at 10 µM, on TXA2, PGF2, and PGH2 production in the incubation media after stimulation with acetylcholine in the presence of L-NAME of aortic rings from WKY or SHR. Values are means ± S.E.M. (n = 7-10). #, p < 0.05 acetylcholine versus basal values; *, p < 0.05 versus WKY; and , p < 0.05 and p < 0.01, respectively, versus acetylcholine-treated endothelium-denuded rings in the absence of phytoestrogens.

Effects of Isoflavones on U46619 [GenBank] -Induced Contractions. The vasoconstrictor response to the TP receptor agonist U46619 [GenBank] was similar in endothelium-denuded aortic rings from SHR and WKY (pD₂ = 8.13 ± 0.03, n = 6 and 7.97 ± 0.07, n = 7, respectively; p > 0.05). Genistein, daidzein, and 17-estradiol (10 and 50 µM) produced a concentration-dependent rightward shift of these curves in both SHR and WKY (Fig. 10).

View larger version (29K): [in this window] [in a new window]   Fig. 10. Effects of genistein, daidzein, and 17-estradiol, at 10 and 50 µM, on the contractions induced by U46619 [GenBank] in endothelium-denuded aortic rings from WKY and SHR. Values are means ± S.E.M. (n = 6-10). *, p < 0.05 and **, p < 0.01 versus vehicle-treated rings.

	Discussion

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Acetylcholine is widely used to assess endothelial function in hypertension and ischemic heart disease (Vanhoutte, 1996). It stimulates muscarinic receptors in endothelial cells and leads to the synthesis and release of several vasoactive factors, including NO, cyclooxygenase-derived vasodilators (e.g., prostacyclin), cyclooxygenase-derived vasoconstrictors (e.g., TXA₂, PGF₂, PGH₂), and endothelium-derived hyperpolarizing factors whose contribution to the endothelium-dependent response varies depending on the vessel studied and experimental conditions. Therefore, the response to acetylcholine (vasodilation or vasoconstriction) depends on the activity of these pathways and the preexisting tone. NO is the most important vasodilator released from the endothelium and in some vessels, such as the rat aorta, endothelium-dependent vasodilation relies almost entirely on the release of NO (Vanhoutte and Miller, 1985). In addition, reduced NO bioavailability is a key event in the pathogenesis of endothelial dysfunction in hypertension and other cardiovascular diseases (Vanhoutte, 1996).

Reduced NO levels in the vessel wall may result from 1) a decrease in NO synthesis and/or 2) an increase in the oxidative inactivation of NO by ROS. Concerning the first issue, changes in aortic NO synthase activity and expression in SHR are controversial and both up-regulation and down-regulation have been found (Chou et al., 1998; Vaziri et al., 1998; Zecchin et al., 2003). In the present study, we found reduced endothelial NO synthase activity in SHR compared with WKY. Interestingly, isoflavones and 17-estradiol increased NO synthase activity in aortic homogenates from both WKY and SHR. However, the contribution of this increase in NO synthase activity to the augmented endothelium-dependent relaxation in SHR is unclear because the increase in NO synthase activity in WKY produced no changes in endothelium-dependent relaxation. Concerning the second issue, among ROS, is critically involved in the breakdown of NO, and endothelial dysfunction in SHR has been associated in many reports with an excess of generation (Suzuki et al., 1995; Cai and Harrison, 2000). In fact, we found that treatment with exogenous superoxide dismutase improves acetylcholine-induced relaxation in SHR rings. In our study, NADPH-stimulated production was increased in SHR aortic rings compared with WKY. This NADPH-stimulated production was strongly reduced by diphenylene iodonium, which inhibits flavin-containing enzymes such as NADPH oxidase, the most important source of in vascular cells (Mohazzab et al., 1994). We also found that genistein, daidzein, and 17-estradiol only reduced NADPH-stimulated aortic production in rings from SHR but not from WKY. These results suggest that genistein and daidzein do not act as scavengers of (Ruiz-Larrea et al., 1997) but rather they specifically reduce the NADPH oxidase overactivity in SHR. Moreover, superoxide dismutase did not increase the endothelium-dependent relaxant responses to acetylcholine in arteries treated with isoflavones or 17-estradiol, suggesting that their inhibitory effect on vascular generation accounts for the improvement of endothelial function in SHR induced by these drugs. Therefore, an increased NO bioavailability in the presence of genistein, daidzein, or 17-estradiol seems to be due to both an increased NO synthesis and a reduced NADPH-dependent production. The latter effect, as well as the increased NO-mediated relaxation induced by acetylcholine, was produced only in SHR. These protective effects are unrelated to an enhanced sensitivity to NO in SHR, since neither isoflavones nor 17-estradiol modified the endothelium-independent relaxant responses induced by the NO donor sodium nitroprusside.

Besides the reduced bioavailability of endothelium-derived NO, the release of endothelium-derived vasoconstrictor factors in response to acetylcholine stimulation is increased in SHR and may counteract the effects of endothelium-derived relaxant factors (Iwama et al., 1992; Ge et al., 1995; Vanhoutte, 1996). The production of this contracting factor(s) involves the activation of endothelial cyclooxygenase-1, and its action the activation on vascular smooth muscle cells of TP receptors (i.e., receptors for TXA₂ and PGH₂) (Yang et al., 2003). Thus, cyclooxygenase inhibitors enhance acetylcholine-induced relaxation and inhibit acetylcholine-induced contractions in SHR (Ge et al., 1995). Endothelium-dependent contractions to acetylcholine in SHR aorta are associated with a greater expression of cyclooxygenase-1, a significant release of PGH₂, and a hypersensitivity of the smooth muscle to this prostanoid, but they are unaffected by thromboxane synthase inhibitors (Ge et al., 1995; Yang et al., 2003). Accordingly, in the present study, acetylcholine induced endothelium-dependent contraction and PGH₂ release (but not TXA₂) in SHR, which were almost absent in WKY. Genistein, daidzein, and 17-estradiol inhibited the endothelium-dependent contractions and the PGH₂ release induced by acetylcholine in SHR. Inhibition of cyclooxygenase-1 (Rosenstock et al., 1997; Wang et al., 2000) activity might be involved in these effects. However, the inhibitory effects of isoflavones were only observed in SHR, suggesting that they do not inhibit cyclooxygenase itself but specifically the cyclooxygenase overactivity observed in SHR. In the SHR aorta, acetylcholine-induced endothelium-dependent contractions have been suggested to involve endothelial superoxide anion and hydrogen peroxide production, which, in turn, activate cyclooxygenase-1 (Yang et al., 2003). Therefore, the inhibitory effect on NADPH-stimulated superoxide production in SHR induced by isoflavones estrogens and 17-estradiol might account for the SHR-specific reduction of endothelial-derived PGH₂.

In addition, these drugs inhibited the contractile response evoked by the TP receptor agonist U46619 [GenBank] in endothelium denuded aortic rings from both strains, which is consistent with results previously reported in several vascular beds from normotensive animals (Martinez et al., 2000; Janssen et al., 2001). The drugs produced a concentration-dependent rightward shift of the curve to U46619 [GenBank] . These effects suggest competitive inhibition of TP receptors, which is consistent with the competitive displacement of [³H]U46619 binding by genistein and daidzein in platelets (Nakashima et al., 1991), even when we cannot rule out other mechanisms interfering with signaling pathways. Thus, genistein, daidzein, and 17-estradiol seem to prevent endothelium-dependent contraction by a double mechanism: inhibiting PGH₂ synthesis at the endothelial level and inhibiting its action in vascular smooth muscle cells.

Genistein and daidzein are structurally very similar, differing only in the substitution at position 5 (a hydroxyl group absent in daidzein), and both show some structural analogy to 17-estradiol. Both isoflavones interact with estrogen receptors with relative selectivity for estrogen receptor over , although genistein is 10 to 100 times more potent than daidzein (Kuiper et al., 1998). Thus, genistein and daidzein could act via binding to estrogen receptors and exerting estrogen-like effects on vascular tissues (Makela et al., 1999). However, despite the similar responses of the isoflavones and 17-estradiol, we could not confirm that classic estrogen receptors mediate these effects because the high-affinity estrogen receptor and pure antagonist ICI 182,780 did not inhibit the effects of genistein, daidzein, or 17-estradiol on acetylcholine-induced relaxation and contraction. This is consistent with reports showing that the improvement of endothelial function in response to acute application of these compounds is unaffected by ICI 182,780 (Karamsetty et al., 2001). Genistein is a well known inhibitor of a broad range of tyrosine kinases (Ogawara et al., 1989). In contrast, daidzein (Ogawara et al., 1989) and 17-estradiol (Von Eye Corleta et al., 1992) lack tyrosine kinase inhibitory activity. The similar profile of all drugs on NO synthase activity, NADPH-stimulated production, PGH₂ release, and U46619 [GenBank] -induced contractions suggests that inhibition of tyrosine kinase is not involved in these effects of genistein.

In conclusion, the isoflavone phytoestrogens genistein and daidzein act like the mammalian estrogen 17-estradiol in restoring endothelial function in SHR, but this effect does not seem to be mediated by estrogen receptor activation or inhibition of tyrosine kinases. An enhanced NO synthase activity and a protective effect on NO from NADPH oxidase-dependent -driven inactivation seem to be involved in the improvement of vascular relaxation to acetylcholine in aortic rings from SHR. Moreover, isoflavones and estradiol inhibited aortic endothelium-dependent contraction to acetylcholine in SHR by reducing the endothelial release of PGH₂ and its vasoconstrictor response induced by activation of TP receptors. These results can explain the mechanisms of the protective effects of these agents on endothelial dysfunction in hypertension.

	Footnotes

 
This work was supported by grants from Ministerio de Educación y Ciencia (SAF 2001-2953 and AGL2004-06685-C04-1/ALI).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.085530.

ABBREVIATIONS: NO, nitric oxide; ROS, reactive oxygen species; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto normotensive rats; DMSO, dimethyl sulfoxide; ICI 182,780, 7,17-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol; L-NAME, N-nitro-L-arginine methyl ester; U46619 [GenBank] , 9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂; PG, prostaglandin; TXA₂, thromboxane A₂.

Address correspondence to: Dr. Juan Duarte, Departemento de Farmacología, Facultad de Farmacia, Campus de Cartuja, Universidad de Granada, 18071 Granada, Spain. E-mail: jmduarte{at}ugr.es

	References

Top Abstract Materials and Methods Results Discussion References

 

Anderson JW, Smith BM, and Washnock CS (1999) Cardiovascular and renal benefits of dry bean and soybean intake. Am J Clin Nutr 70 (Suppl): 464S-474S.[Abstract/Free Full Text]

Auch-Schwelk W, Katusic ZS, and Vanhoutte PM (1990) Thromboxane A₂ receptor antagonists inhibit endothelium-dependent contractions. Hypertension 15: 699-703.[Abstract/Free Full Text]

Bermejo A, Zarzuelo A, and Duarte J (2003) In vivo vascular effects of genistein on a rat model of septic shock induced by lipopolysaccharide. J Cardiovasc Pharmacol 42: 329-338.[CrossRef][Medline]

Cai H and Harrison DG (2000) Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840-844.[Abstract/Free Full Text]

Chou T-C, Yen M-H, Li C-Y, and Ding Y-A (1998) Alterations of nitric oxide synthase expression with aging and hypertension in rats. Hypertension 31: 643-648.[Abstract/Free Full Text]

Duarte J, Perez-Palencia R, Vargas F, Ocete MA, Perez-Vizcaino F, Zarzuelo A, and Tamargo J (2001) Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol 133: 117-124.[CrossRef][Medline]

Ge T, Hughes H, Junquero DC, Wu KK, Vanhoutte PM, and Boulanger CM (1995) Endothelium-dependent contractions are associated with both augmented expression of prostaglandin H synthase-1 and hypersensitivity to prostaglandin H₂ in the SHR aorta. Circ Res 76: 1003-1010.[Abstract/Free Full Text]

Honore EK, Williams JK, Anthony MS, and Clarkson TB (1997) Soy isoflavones enhance coronary vascular reactivity in atherosclerotic female macaques. Fertil Steril 67: 148-154.[CrossRef][Medline]

Iwama Y, Kato T, Muramatsu M, Asano H, Shimizu K, Toki Y, Miyazaki Y, Okumura K, Hashimoto H, and Ito T (1992) Correlation with blood pressure of the acetylcholine-induced endothelium-derived contracting factor in the rat aorta. Hypertension 19: 326-332.[Abstract/Free Full Text]

Janssen LJ, Lu-Chao H, and Netherton S (2001) Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho kinase. Am J Physiol 280: L666-L674.

Karamsetty MR, Klinger JR, and Hill NS (2001) Phytoestrogens restore nitric oxide-mediated relaxation in isolated pulmonary arteries from chronically hypoxic rats. J Pharmacol Exp Ther 297: 968-974.[Abstract/Free Full Text]

Kitayama J, Kitazono T, Ooboshi H, Ago T, Ohgami T, Fujishima M, and Ibayashi S (2002) Chronic administration of a tyrosine kinase inhibitor restores endothelial functional and morphological changes of the basilar artery during chronic hypertension. J Hypertens 20: 2205-2211.[CrossRef][Medline]

Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B, and Gustafsson JA (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139: 4252-4263.[Abstract/Free Full Text]

Kurzer MS and Xu X (1997) Dietary phytoestrogens. Annu Rev Nutr 17: 353-381.[CrossRef][Medline]

Lindner V, Kim SK, Karas RH, Kuiper GG, Gustafsson JA, and Mendelsohn ME (1998) Increased expression of estrogen receptor-beta mRNA in male blood vessels after vascular injury. Circ Res 83: 224-229.[Abstract/Free Full Text]

Makela S, Savolainen H, Aavik E, Myllarniemi M, Strauss L, Taskinen E, Gustafsson JA, and Hayry P (1999) Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors alpha and beta. Proc Natl Acad Sci USA 96: 7077-7082.[Abstract/Free Full Text]

Manach C, Scalbert A, Morand C, Remesy C, and Jimenez L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79: 727-747.[Abstract/Free Full Text]

Martinez MC, Randriamboavonjy V, Ohlmann P, Komas N, Duarte J, Schneider F, Stoclet JC, and Andriantsitohaina R (2000) Involvement of protein kinase C, tyrosine kinases and Rho kinase in Ca²⁺ handling of human small arteries. Am J Physiol 279: H1228-H1238.

Mitchell JA, Sheng H, Forstermann U, and Murad F (1991) Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle. Br J Pharmacol 104: 289-291.[Medline]

Mohazzab KM, Kaminski PM, and Wolin MS (1994) NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium. Am J Physiol 266: H2568-H2572.

Nakashima S, Koike T, and Nozawa Y (1991) Genistein, a protein tyrosine kinase inhibitor, inhibits thromboxane A₂-mediated human platelet responses. Mol Pharmacol 39: 475-480.[Abstract]

Ogawara H, Akiyama T, Watanabe S, Ito N, Kobori M, and Seoda Y (1989) Inhibition of tyrosine protein kinase activity by synthetic isoflavones and flavones. J Antibiot 42: 340-343.[Medline]

Ohara Y, Peterson TE, and Harrison DG (1993) Hypercholesterolemia increases endothelial superoxide anion production. J Clin Investig 91: 2546-2551.

Orshal JM and Khalil RA (2004) Gender, sex hormones and vascular tone. Am J Physiol 286: R233-R249.

Rosenstock M, Danon A, and Rimon G (1997) Prostaglandin H synthase: protein synthesis-independent regulation in bovine aortic endothelial cells. Am J Physiol 273: C1749-C1755.

Ruiz-Larrea MB, Mohan AR, Paganga G, Miller NJ, Bolwell GP, and Rice-Evans CA (1997) Antioxidant activity of phytoestrogenic isoflavones. Free Radic Res 26: 63-70.[Medline]

Russell KS, Haynes MP, Sinha D, Clerisme E, and Bender JR (2000) Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci USA 97: 5930-5935.[Abstract/Free Full Text]

Schachinger V, Britten MB, and Zeiher AM (2000) Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation 101: 1899-1906.[Abstract/Free Full Text]

Simons LA, von Konigsmark M, Simons J, and Celermajer DS (2000) Phytoestrogens do not influence lipoprotein levels or endothelial function in healthy, postmenopausal women. Am J Cardiol 85: 1297-1301.[CrossRef][Medline]

Squadrito F, Altavilla D, Morabito N, Crisafulli A, D'Anna R, Corrado F, Ruggeri P, Campo GM, Calapai G, Caputi AP, et al. (2002) The effect of the phytoestrogen genistein on plasma nitric oxide concentrations, endothelin-1 levels and endothelium dependent vasodilation in postmenopausal women. Atherosclerosis 163: 339-347.[CrossRef][Medline]

Squadrito F, Altavilla D, Squadrito G, Saitta A, Cucinotta D, Minutoli L, Deodato B, Ferlito M, Campo GM, Bova A, et al. (2000) Genistein supplementation and estrogen replacement therapy improve endothelial dysfunction induced by ovariectomy in rats. Cardiovasc Res 45: 454-462.[Abstract/Free Full Text]

Suzuki H, Swei A, Zweifach BW, and Schmid-Schonbein GW (1995) In vivo evidence for microvascular oxidative stress in spontaneously hypertensive rats. Hydroethidine microfluorography. Hypertension 25: 1083-1089.[Abstract/Free Full Text]

Vanhoutte PM (1996) Endothelial dysfunction in hypertension. J Hypertens 14: S83-S93.

Vanhoutte PM and Miller VM (1985) Heterogeneity of endothelium-dependent responses in mammalian blood vessels. J Cardiovasc Pharmacol 7: S12-S23.

Vaziri ND, Ni Z, and Oveisi F (1998) Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31: 1248-1254.[Abstract/Free Full Text]

Von Eye Corleta H, Strowitzki T, Kellerer M, and Haring HU (1992) Insulin-like growth factor I-dependent tyrosine kinase activity in stromal cells of human endometrium in vitro. Am J Physiol 262: E863-E868.

Wakeling AE, Dukes M, and Bowler J (1991) A potent specific pure antiestrogen with clinical potential. Cancer Res 51: 3867-3873.[Abstract/Free Full Text]

Wang H, Nair MG, Strasburg GM, Booren AM, Gray I, and Dewitt DL (2000) Cyclooxygenase active bioflavonoids from Balaton tart cherry and their structure activity relationships. Phytomedicine 7: 15-19.[Medline]

Yang D, Feletou M, Levens N, Zhang JN, and Vanhoutte PM (2003) A diffusible substance(s) mediates endothelium-dependent contractions in the aorta of SHR. Hypertension 41: 143-148.[Abstract/Free Full Text]

Zecchin HG, Bezerra RM, Carvalheira JB, Carvalho-Filho MA, Metze K, Franchini KG, and Saad MJ (2003) Insulin signalling pathways in aorta and muscle from two animal models of insulin resistance-the obese middle-aged and the spontaneously hypertensive rats. Diabetologia 46: 479-491.[Medline]

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