See page 1986 OBJECTIVES BACKGROUND METHODS RESULTS CONCLUSIONS

Transcription

1 Journal of the American College of Cardiology Vol. 35, No. 7, by the American College of Cardiology ISSN /00/$20.00 Published by Elsevier Science Inc. PII S (00) Plant-Derived Estrogens Relax Coronary Arteries In Vitro by a Calcium Antagonistic Mechanism Gemma A. Figtree, MB, BMEDSCI,* Huw Griffiths, MB, BSC, Ying-Qing Lu, PHD, Carolyn M. Webb, PHD, Kenneth MacLeod, PHD, Peter Collins, MD, FRCP, FACC* London, United Kingdom and Sydney, Australia OBJECTIVES BACKGROUND METHODS RESULTS CONCLUSIONS To investigate the potential for plant derived estrogens (phytoestrogens) genistein, phloretin, biochanin A and zearalanone to relax rabbit coronary arteries in vitro and to determine the mechanism(s) of such relaxation. Epidemiological data suggests a reduction in the incidence of coronary heart disease in humans who have a high intake of phytoestrogens. Isolated rabbit coronary artery rings were suspended in individual organ baths, precontracted with potassium chloride (30mM), and the relaxing effects and mechanisms of relaxation to genistein, phloretin, biochanin A and zearalanone were determined by measurement of isometric tension. Genistein, phloretin and biochanin A induced significant gender-independent relaxation in rings with and without endothelium. Inhibition of nitric oxide and prostaglandin synthesis with L-NAME and indomethacin had no effect on genistein-induced relaxation. Relaxation was unaffected by the specific estrogen receptor antagonist ICI 182,780, the ATP-sensitive potassium channel inhibitor glibenclamide and the potassium channel inhibitor, barium chloride. Calcium concentration-dependent contraction curves in high potassium depolarization medium were significantly shifted to the right and downward after incubation with genistein and zearalanone. An inhibitory effect of genistein (2 M) on L-type calcium current in guinea-pig ventricular myocytes confirmed a calcium antagonist relaxing mechanism of action. In healthy volunteers, plasma genistein levels of approximately 2 M are achieved after ingestion of a commercially available soy protein drink (Supro) containing 37 mg genistein. This study demonstrates that phytoestrogens induce endothelium-independent relaxation of coronary arteries; the mechanism involves calcium antagonism. These mechanisms may contribute to the potential long-term cardiovascular protective effect of these substances. (J Am Coll Cardiol 2000;35: ) 2000 by the American College of Cardiology Phytoestrogens are synthesized by all plants, particularly soy beans, and possess estrogenic activity in animals (1 6). Phytoestrogens are naturally found in many foods and they are defined as plant substances that are structurally or functionally similar to estradiol. They consist of a number of classes, including lignans, isoflavones, coumestans and resorcyclic acid lactones. Lignans and isoflavones have been detected in humans and both have been shown to have See page 1986 From the Cardiac Medicine, Imperial College School of Medicine at the National Heart & Lung Institute, London, United Kingdom and *Department of Physiology, University of Sydney, Sydney, Australia. This study was supported, in part, by a grant from Protein Technologies International, St Louis, Missouri. Manuscript received December 30, 1998; revised manuscript received December 30, 1999, accepted February 21, biological activity in man and animals (7). The human diet provides the precursors for the formation of mammalian ligans and isoflavones. The highest concentration of ligans are found in oil seeds, cereal bran, whole cereals, vegetables, legumes and fruits (8). The isoflavones are less widely distributed in plant foods, but they do occur in high concentration in soy beans, chick peas and other legumes as well as toothed medic, bluegrass and clovers (9). A more complete review of the clinical effects of phyto-oestrogens has been published (10). Lignans and isoflavones are derived from dietary precursors largely by gut flora. The major isoflavones found in humans, namely diadzein and genistein are derived from formonentin and biochanin A, respectively. As well as evidence that ovarian estrogens have cardiovascular protective effects (11,12), there is evidence that dietary phytoestrogens may also confer cardiovascular protection. Epidemiological data suggest a reduction in the

2 1978 Figtree et al. JACC Vol. 35, No. 7, 2000 Plant Estrogens and Coronary Arteries June 2000: Abbreviations and Acronyms ACh acetylcholine HEPES hydroxyethyl pipirazine-ethanesulphonic acid I Ca L-type calcium current L-NAME N omega -nitro-l-arginine methyl ester incidence of coronary heart disease in humans who have a high intake of phytoestrogens (13,14). Increased plasma levels of the phytoestrogen genistein are suggested as an explanation for the infrequency of hot flashes and menopausal symptoms in Japanese women (15). There are structural similarities between the steroidal nucleus of 17beta-estradiol and the rigid ring structure of phytoestrogens. Flavanoids are compounds containing a characteristic aromatic trimeric heterocyclic nucleus, usually occurring in glycosidic form and widely distributed in plants. Using competitive binding techniques between 17beta-[ 3 H] estradiol and the estrogen receptor in cell-free extracts, it was shown that hydroxylated flavanoids (including chalones [isoliquiritigenin, phloretin], isoflavones [genistein], flavones [apipenin] and flavonones) interact directly with the estrogen receptor (16). Genistein has vascular activity and can attenuate acetylcholine (ACh)- induced coronary vasoconstriction in atherosclerotic cynomolgus monkeys in a very similar way to estrogen (17), which may imply a stimulatory effect on the nitric oxide pathway. The mechanism involved in coronary artery vasodilation may also contribute to the cardioprotective effects of estrogen (18 21). Despite the increasing interest in the effects of phytoestrogens on the cardiovascular system, it is unknown whether they share the coronary artery vasodilator properties of estrogen. We, therefore, investigated the vascular effects of the phytoestrogens genistein, phloretin, biochanin A and zearalanone in rabbit coronary arteries and isolated cardiac myocytes in vitro. METHODS Animals and tissues. Adult male or nonpregnant female New Zealand white rabbits (2.5 3 kg) were killed by an overdose of pentobarbitone (60 mg kg 1 and heparin (150 U kg 1 ). The heart was removed and epicardial coronary arteries were dissected free of connective tissue. Arterial rings were prepared and, in some rings, the endothelium was removed by gentle rubbing with a wooden probe. Each ring (2 3 mm length) was suspended horizontally between two stainless steel parallel hooks for the measurement of isometric tension in individual organ baths containing 10 ml modified Krebs solution at 37 C, bubbled with 95% O 2 and 5% CO 2. The composition of modified Krebs solution was as follows (mm): NaCl, 118.3; KCl, 4.7; CaCl 2, 2.5; MgSO 4, 1.2; K 2 PO 4, 1.2 and glucose, Coronary arterial rings with or without endothelium from male and female rabbits were allowed to stabilise for 90 min under a resting tension of 1 g (9.8 mn) before being contracted. Preparations were exposed to maximally effective concentrations of a contractile agonist (K,30mM)to ensure stabilization of the rings. The agonist was then removed and the ring reequilibrated. The presence or absence of endothelium was always verified by observing the relaxation response to acetylcholine (ACh). Effect of phytoestrogens on precontracted coronary arteries. Coronary arterial rings with or without endothelium were contracted with K (30 mm). Increasing concentrations of the phytoestrogens genistein (10, 20 and 40 M), phloretin (5, 10, 20 and 40 M) or biochanin A (3, 10 and 30 M) were added at half-log increments at the plateau of the previous response. The response at each concentration of phytoestrogen was measured. Simultaneous timematched ethanol control curves were constructed using an equivalent volume of solvent as that used to dissolve the phytoestrogen. Comparative relaxing effects of genistein in the presence of a variety of inhibitors. In experiments where specific inhibitors were used, they were added 20 min before precontarction with 30 mm KCl. Responses to genistein (1 40 M) were then measured. The inhibitors tested were N omega -nitro- L-arginine methyl ester (L-NAME 0.1 mm) an inhibitor of NO synthesis from L-arginine in vascular endothelial cells (22) and indomethacin (10 M), an inhibitor of prostanoid synthesis (23). To examine the possible role of potassium conductance on flavanoid-induced coronary relaxation, glibenclamide (3 M), an ATP-sensitive potassium channel inhibitor, and barium chloride, a nonspecific inhibitor of potassium channels (24). To examine the possible role of the classical estrogen receptor in mediating phytoestrogeninduced relaxation, rings with and without endothelium were incubated in the specific estrogen receptor antagonist ICI 182,780 (25) (10 M). Rings without endothelium were incubated in methylene blue (10 M) to determine the role of cgmp in the relaxant effect of genistein (26). Effect of phytoestrogens on calcium concentration responses in rabbit coronary arteries. Rabbit coronary arterial rings without endothelium were incubated in calciumfree solution containing 0.5 mm EGTA for 10 min. The calcium concentration-dependent contraction curves were then performed in high K depolarization medium (100 mm). Rings were readjusted in modified Krebs for 20 min and then incubated in calcium-free solution containing EGTA (0.5 mm) for a further 10 min. Subsequently rings were incubated with genistein (20 and 40 M) or zearalanone (1 and 10 M). The calcium concentrationdependent contraction curves were then repeated. Comparative relaxing effects of phytoestrogens on precontracted coronary arteries. The relaxing effects of an equal concentration (10 M) of genistein, phloretin and

3 JACC Vol. 35, No. 7, 2000 June 2000: Figtree et al. Plant Estrogens and Coronary Arteries 1979 biochanin A on coronary arterial rings contracted with K (30 mm) was determined. Electrophysiological experiments. Electrophysiological experiments were performed on isolated left ventricular myocytes from adult male guinea-pigs prepared by enzymatic dissociation as previously described (27). Animals were heparinized and killed by cervical dislocation, the heart rapidly removed, the aorta cannulated and the heart retrogradely perfused on a Langendorff apparatus with normal Tyrode solution at 37 C before enzymatic digestion. The composition of normal Tyrode was as follows (mm): NaCl 140, KCl 6, CaCl 2, MgCl 1, Glucose 10, hydroxyethylpipirazine-ethanesulphonic acid (HEPES) 10, titrated to ph 7.4 with 2M NaOH. The isolated cells were stored in Dulbecco s medium (Gibco) at room temperature. Cells were allowed to settle for up to 5 min on the glass base of a low volume (20 l) plexiglass superfusion chamber mounted on the stage of an inverted microscope after applying 1 l mouse laminin (Gibco) to the base of the chamber. Normal Tyrode (NT) at room temperature was carried to the chamber by means of gas impermeable tubing at 2.5 ml/min, and at this rate solution switching was complete within 4 s. Genistein was added from a 40 mm stock solution in ethanol. Borosilicate glass microelectrodes with resistances of 20 to 35 M omego to minimize intracellular dialysis were filled with a solution of 2M KCl, 5 mm HEPES and 100 nm EGTA at ph 7.2. Electrophysiological recordings were made with an Axoclamp-2A amplifier controlled by pclamp6 software, which was also used for data analysis (Axon Instruments). Action potentials were recorded by stimulating the cells in current clamp mode with a 1 na pulse of 10 ms duration. Voltage clamp experiments were performed in switch clamp mode (discontinuous single electrode voltage clamp, 5 6 khz) with gains between 0.3 and 0.8 na/mv, maximized to obtain sharp voltage steps without oscillation. Membrane current was filtered at 0.1 khz, digitized at 5 khz and analyzed as the average of five consecutive recordings. L-type calcium current (I Ca ) was measured as the difference between the nifedipine sensitive, time-dependent peak inward current and the steady-state current present at the end of a 400 ms clamp step. Cell shortening was measured at one cell edge with a video detection system. Human volunteer study to determine plasma levels of ingested genistein. Fourteen male volunteers ingested a commercially available isolated soy protein milk beverage Supro or placebo (Protein Technologies International, St. Louis, Missouri) on three occasions, on day one at 7:00 AM and 7:00 PM and on day two at 7:00 AM. A blood sample was taken on day two at 11:00 AM, 4 h after the ingestion of the last soy protein drink. The volunteers were asked not to consume soy protein containing foods, such as tofu or drinks such as beer during the time of the study and for 24 h before the first ingestion. Supro contains 60 mg of isoflavones, 37 mg of which is genistein. The mucosal lining of the gut Figure 1. Bar graph showing the relaxing effect of genistein (10, 20 and 40 M) on rabbit coronary artery rings with (n 6) and without endothelium (n 6). Rings were precontracted with K (30 mm). Data are expressed as percentage relaxation of contraction induced by K (mean SEM). Control indicates a timematched equivalent volume of solvent (n 12 14). *indicates significant differences in comparison with control; * p 0.05; ** p 0.01; *** p is necessary for the activation of genistein in this form and activation takes approximately 3 to 5 h. The study was a randomized, double blind study and was placebo-controlled. The placebo contained no phytoestrogens. Blood samples were taken before the first ingestion and 4 h after the third ingestion of either placebo or active Supro. Plasma levels of genistein were measured by isotope dilution gas chromatography-mass spectroscopy by a group experienced in these measurements (Dr. M. Morton, Tenovus Cancer Research Center, University of Wales College of Medicine, Cardiff, United Kingdom). Drugs. The following drugs were used: genistein (Sigma soy source); phloretin (Sigma); biochanin A (Sigma); zearalanone (Sigma); N omega -nitro-l-arginine methyl ester (Sigma); indomethacin (Sigma); glibenclamide (ICN); ICI 182,780 (a gift from Zenica Pharma, Cheshire, United Kingdom); Supro or placebo (Protein Technologies International, St Louis, Missouri). Data analysis. All results are expressed as mean SEM. Relaxation is expressed as percentage relaxation of contraction induced by K (30 mm). The results were analyzed with analysis of variance, and the Student-Newman-Keuls test was used for multiple comparisons. A probability level of less than 0.05 was considered significant. The number of animals is indicated by n, unless indicated otherwise. RESULTS Relaxing effect of phytoestrogens on precontracted coronary arteries. K (30 mm) induced significant contraction, comparable in rings with or without endothelium ( and mn; p 0.05). Genistein (Figs. 1 and 4), phloretin (Fig. 2) and biochanin A (Fig. 3)

4 1980 Figtree et al. JACC Vol. 35, No. 7, 2000 Plant Estrogens and Coronary Arteries June 2000: Figure 4. Original tracing demonstrating genistein (10, 20 and 40 M)-induced relaxation in K -contracted coronary arterial rings. Figure 2. Bar graph showing the relaxing effect of phloretin (5, 10, 20 and 40 M) on rabbit coronary artery rings with (n 6) and without (n 6) endothelium. Rings were precontracted with K (30 mm). Data are expressed as percentage relaxation of contraction induced by K (mean SEM). Control indicates a timematched equivalent volume of solvent (n 12 14). *indicates significant differences in comparison with control; * p 0.05; *** p induced significant relaxation of contracted rings compared with time-matched controls in a concentration-dependent manner. There were no significant differences in relaxation between arteries from male or female rabbits (n 6; p 0.05) or between rings with or without endothelium (n 6; p 0.05). The same concentration (10 M, n 6) of phytoestrogens relaxed precontracted coronary arteries with different potencies. The order of potency was biochanin A phloretin genistein (Fig. 5). Effect of the presence of a variety of inhibitors on genistein-induced relaxation. Incubation with the inhibitor of NO synthesis, L-NAME, or the inhibitor of prostanoid synthesis, indomethacin, (all n 6) did not inhibit relaxation induced by genistein in rabbit coronary arterial rings with endothelium (Fig. 6a). Incubation in ICI 182,780, an estrogen-receptor antagonist, did not affect the relaxation induced by genistein in rings with endothelium (Fig. 6a). Blockade of the ATP-sensitive potassium channel inhibitor glibenclamide and the nonspecific inhibitor of potassium channels barium chloride had no effect on relaxation induced by genistein in rings without endothelium (Fig. 6b). Methylene blue had no effect on relaxation induced by genistein, phloretin or biochanin A in rings without endothelium (Fig. 6b). Calcium antagonistic effects of genistein and zearalanone on rabbit coronary arterial rings. The calcium concentration-dependent contraction curves in high K (100 M) depolarization medium were shifted to the right after incubation with genistein (20 and 40 M, n 6) and zearalanone (1 and 10 M, n 6) in rings without endothelium compared with control. Maximal contraction was also reduced (Fig. 7, a and b). Inhibitory effect of genistein on I Ca. To further investigate the effects of phytoestrogens on calcium dependent contraction, I Ca was measured directly in calcium tolerant, healthy guinea-pig ventricular myocytes with resting membrane potentials of 70 mv isolated from six hearts. Cells were held under voltage clamp at 40 mv to inactivate fast sodium current and calcium current elicited by applying 400 ms clamp steps to 0 mv at a frequency of 0.5 Hz. Results are demonstrated in Figure 8. Panel A demon- Figure 3. Bar graph showing the relaxing effect of biochanin A (3, 10 and 30 M) on rabbit coronary artery rings with (n 6) and without endothelium (n 6). Rings were precontracted with K (30 mm). Data are expressed as percentage relaxation of contraction induced by K (mean SEM). Control indicates a timematched equivalent volume of solvent (n 12 14). *indicates significant differences in comparison with control; * p 0.05; ** p Figure 5. Comparison of the relaxant effects of equivalent concentrations (10 M) of genistein, phloretin and biochanin A.

5 JACC Vol. 35, No. 7, 2000 June 2000: Figtree et al. Plant Estrogens and Coronary Arteries 1981 Figure 6. (a) The effect of incubation in L-NAME (L-NA), ICI 182,780 (ICI) and indomethacin (Indometh) on genistein-induced relaxation in rings with endothelium compared with control rings (no incubation) (n 6, p 0.05). (b) The effect of incubation in barium chloride (BaCl), glibenclamide (Gliben) and methylene blue (Methyl blue) on genistein-induced relaxation in rings without endothelium compared with control rings (no incubation) (n 6, p 0.05). strates the reduction in steady state peak I Ca accompanied by reduced cell contraction, 1 min after superfusion with 2 M genistein. Peak I Ca was found to be reduced from pa to pa (p 0.001, n 12) with a reduction in cell contraction from m to m (p 0.001, n 12). Current-voltage and contraction-voltage curves were constructed with 400 ms clamp steps to varying potentials from a holding potential of 40 mv at a frequency of 0.5 Hz as shown in panel B. Inhibition of I Ca occurred over the range of potentials tested, but the shape of the curve was not affected by genistein with peak I Ca and contraction occurring between 0 and 10 mv. The effect of genistein was dose-dependent with, most notably, complete abolition of isolated cell contraction at the highest concentration of 40 M. The histograms in panel C were constructed by superfusing with increasing concentrations of genistein and measurements made at steady state, 90 s after each solution switch. Reversibility of genistein effect. On returning to superfusion with NT and with continued stimulation under voltage clamp steps, there was partial recovery of I Ca and cell contraction over the first minute to pa (p 0.05) and m (p 0.01), respectively (n 11). No further recovery was observed over an additional 3 min. The original trace in panel A was taken from one of a series of experiments where peak I Ca was remeasured after a train of stimulated action potentials in current clamp mode after washout of genistein. Under these conditions, I Ca Figure 7. The effect of genistein (Ge: 20 and 40 M) (a) and zearalenone (Zea; 1 and 10 M) (b) on the calcium concentrationdependent contraction curves in rabbit coronary arteries without endothelium compared with control. Data are expressed as percentage of maximal contraction induced by calcium in controls (mean SEM). *indicates significant differences in comparison with corresponding controls; * p 0.05; ** p 0.01; *** p demonstrated full recovery to (p 0.8, compared with control, n 4) with recovery of cell contraction to m (control: m, p 0.27, n 4). Human volunteer study plasma genistein levels. Seven volunteers (age 33 4 years) received placebo and seven volunteers (age 42 5 years, NS placebo vs. active) received the active Supro drink. In the seven volunteers who received placebo, the baseline genistein levels were 0.02 M 0.01 M, and after placebo the levels were 0.04 M 0.05 M (p NS). In the active Supro group, the baseline genistein levels were 0.03 M 0.02 M. After receiving active Supro, the genistein levels were 2.1 M 0.8 M (p , placebo vs. active Supro). DISCUSSION We have shown that the phytoestrogens genistein, phloretin, biochanin A and zearalanone induce significant relaxation of coronary artery rings. Relaxation was dosedependent. No difference was observed between rings with or without endothelium or between rings from male versus those from female rabbits. Preincubation with L-NAME, indomethacin, glibenclamide, barium chloride or methylene blue did not affect genistein-induced coronary artery relax-

6 1982 Figtree et al. JACC Vol. 35, No. 7, 2000 Plant Estrogens and Coronary Arteries June 2000: Figure 8. The effects of genistein on calcium current and contraction of isolated guinea-pig cardiac myocytes. (A) Averaged traces from a representative cell on repetitive step depolarizations from a holding potential of 40 mv to 0 for 400 ms. (i) Control in normal Tyrode, (ii) after 1 min stabilization in 2 M genistein, (iii) after washout in normal Tyrode and a period of action potential stimulation under current clamp. (B) (i) Current-voltage and (ii) contraction-voltage relationships in normal Tyrode (open circle) and 2 M genistein (solid circle) with step depolarizations of 400 ms from a holding potential of 40 mv (n 9 unless indicated otherwise). (C) Dose-response relationships for (i) L-type calcium current and (ii) contraction after 90 s in varying concentrations of genistein (n indicated for each bar). In all cases, data are expressed as mean SEM. * indicates p 0.05; ** indicates p 0.01; *** indicates p compared with control. ation. Similarly, the specific estrogen receptor antagonist ICI 182,780 did not affect the coronary relaxation induced by genistein. Incubation with both genistein and zearalanone shifted the calcium concentration-dependent contraction curve to the right and reduced the maximal contraction.

7 JACC Vol. 35, No. 7, 2000 June 2000: Figtree et al. Plant Estrogens and Coronary Arteries 1983 Calcium antagonist mechanism of relaxation. The finding that incubation with genistein or zearalenone shifted the calcium concentration-dependent contraction curve to the right in high K depolarization medium suggests that these phytoestrogens are acting via a calcium antagonistic mechanism to cause relaxation of coronary arteries. This is similar to estrogen and progesterone (18,28,29). Influx of calcium is known to be an important mediator of excitationcontraction coupling in smooth muscle cells. Thus, the inhibition of calcium influx may be an important mechanism of the action of genistein. Though the maximal contraction curves to the right in a parallel fashion would suggest that phytoestrogens interfere with calcium influx through the voltage-operated calcium channel. The most potent relaxation was induced by biochanin A followed by phloretin then genistein. The mechanism of this differential effect on coronary artery relaxation was not determined, but a structure-function relationship is a possibility. A calcium antagonist action of genistein was proven by demonstrating that it reversibly inhibited L-type calcium current and contraction in isolated guinea-pig ventricular myocytes in a concentration-dependent manner. We have previously shown a similar effect of 17beta-estradiol (28). At 40 M genistein, the highest concentration evaluated, isolated cell contraction was abolished as the result of inhibition of peak I Ca to 6% of control. This mirrored the total relaxation of precontracted coronary rings exposed to the same concentration. These results are consistent with a calcium antagonistic mechanism for phytoestrogen-induced vasodilatation. Importantly, the degree of I Ca inhibition observed with 2 M genistein is comparable with that likely to be occurring with calcium antagonists at clinically relevant doses (30,31). Complete recovery of I Ca did not occur until membrane potential was restored and action potentials were stimulated. This might reflect analogous behavior to that of calcium antagonists whose affinity for the inactivated state of the L-type channel exceeds that for the resting state. At 40 mv, 20% of the channels remain inactivated, thus delaying dissociation of genistein (31). This question has not been directly addressed for phytoestrogens, but the relationship holds true for 17beta-estradiol (28). Other potential mechanisms of vascular relaxation by phytoestrogens. The phytoestrogen genistein is a tyrosine kinase inhibitor (32 34). The concentrations of genistein that induced vascular smooth muscle relaxation are within the inhibitor concentration values for genistein against tyrosine protein kinases ( M). However, the relaxant effect of genistein via inhibition of tyrosine kinase activity depends very much on the specific agonist used to induce contraction (35 37). Tyrosine kinase inhibitors reversibly inhibit alpha-adrenergic and muscarinic receptormediated contraction. In contrast, genistein only causes slight inhibition of contraction elicited by potassiuminduced depolarization in guinea pig taenia coli, mesenteric microvessels and canine carotid arteries. Thus, the demonstration of almost complete relaxation ( 99%) in this study in coronary arteries precontracted by depolarization with K (30 mm) suggests that mechanisms other than tyrosine kinase inhibition are responsible for coronary artery relaxation in this preparation. The concentrations of genistein that have been shown to relax coronary arteries and inhibit calcium current in isolated myocytes (2 M) in this study are equivalent to plasma concentrations measured in the volunteer study. These data, therefore, support the fact that the mechanisms demonstrated in vitro may result in cardiovascular actions in vivo in humans. Neither endothelium-derived nor prostaglandins appear to be involved in the coronary artery relaxing effect of phytoestrogens because their respective inhibitors, namely L-NAME and indomethacin, had no effect on the relaxing response to genistein. The classical estrogen receptor (ERalpha) has been found in arterial smooth muscle cells, including coronary arteries, of several mammalian species (38,39). Phytoestrogens have been shown to interact with the ERalpha (4,16,40) although this has not been demonstrated in rabbit coronary arteries. The possible involvement of ERalpha in genisteininduced coronary artery relaxation was tested using the specific ER antagonist ICI 182,780 (7 alpha-[9-(4,4,5,5,- pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10)-triene- 3,17beta-diol) (25). The lack of inhibition of ICI 182,780 of the relaxant effects of genistein suggests that the ERalpha does not play a role in the acute vasorelaxant effects of genistein in coronary arteries in vitro. Our data support a non-receptor mechanism of relaxation of vascular smooth muscle by genistein. However, our data do not preclude actions of dietary genistein (plasma levels approximate 1 M [41]) on the ER, for which it has a relatively high affinity (K i 0.1 M) (40). An ER-mediated mechanism could still contribute to the long-term cardioprotective effect of this phytoestrogen in vivo (13). Recently an estrogen receptor-beta (ERbeta) was identified in rat prostate tissue (42). This receptor has also been identified in vascular tissue and may be responsible for some vascular effects of estrogen. Similar effects are possible for phytoestrogens (43). In our experiments a steroid receptormediated effect through ERbeta cannot be excluded. Activation or inhibition of potassium channel activity in arterial smooth muscle membrane contributes to regulation of membrane potential and provides an important mechanism involved in dilatation or constriction of arteries (44). In our study, incubation with the ATP-sensitive potassium channel blocker glibenclamide (45) and the nonspecific potassium channel blocker barium chloride did not affect genistein-induced relaxation, which suggests that potassium channels do not play a role in this relaxation. However the significance of these results may be limited by the fact that the rings were precontracted with potassium chloride. The demonstration of relaxing effects of genistein in coronary arteries may have important implications in pa-

8 1984 Figtree et al. JACC Vol. 35, No. 7, 2000 Plant Estrogens and Coronary Arteries June 2000: tients with coronary artery disease and myocardial ischemia. Being a non-sex steroidal estrogen, the benefits in the vascular system may be achieved without significant sexual effects. Genistein shares other cardiovascular beneficial actions with estrogen. The effects of genistein are tissuespecific, with estrogen agonist effects on plasma lipid concentrations, plasma lipoprotein concentrations (46,47) and preservation of bone mass (48,49) that are similar in magnitude to mammalian estrogens but without estrogenic effects on the uterus at these same doses (50,51). They may also confer cardiovascular protection to women after menopause, without the associated increased risk of uterine and breast cancer that are associated with estrogen administration. The lack of sex effects, as well as the lack of difference in response between coronary arteries from men and women, means that the benefits conferred by genistein may potentially apply to men. Conclusions. We have shown that the phytoestrogens genistein, phloretin, biochanin A and zearalanone induce significant endothelium-independent relaxation in isolated rabbit coronary arteries, which is independent of sex and of the classical estrogen receptor. An important aspect of this work is that the concentrations that have been shown to induce coronary artery relaxation are in the same range as the plasma concentrations of phytoestrogens measured in the plasma of volunteers taking a commercially available soy protein drink, namely Supro. The relaxation induced by genistein is neither mediated by release of vasodilator, prostanoids or cyclic GMP. ATP-sensitive potassium channels are probably not involved in the mechanism of genistein-induced relaxation. The mechanism may involve calcium-antagonism as observed for genistein and zearalanone and proven with electrophysiological experiments in isolated myocytes for genistein. The demonstrated vasorelaxation of coronary arteries induced by phytoestrogens may be an important finding, as the mechanism involved in the relaxing response may be linked to the decreased cardiovascular risk in populations with high dietary phytoestrogen intake. Reprint requests and correspondence: Dr. Peter Collins, Cardiac Medicine, Imperial College School of Medicine at the National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom. peter.collins@ic.ac.uk. REFERENCES 1. Farnsworth NR, Bingel AS, Cordell GA, Crane FA, Fong HH. Potential value of plants as sources of new antifertility agents I. J Pharm Sci 1975;64: Farnsworth NR, Bingel AS, Cordell GA, Crane FA, Fong HS. Potential value of plants as sources of new antifertility agents II. J Pharm Sci 1975;64: Farmakalidis E, Hathcock JN, Murphy PA. Oestrogenic potency of genistin and daidzin in mice. Food Chem Toxicol 1985;23: Martin PM, Horwitz KB, Ryan DS, McGuire WL. Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology 1978;103: Phytochemistry. In: Harbone JB. Miller, LP, editors. Flavanoids. New York: Van Nostrand Reinhold 1973: Harbone JB. Phytochemical methods. Phenolic Compounds. London: Chapman and Hall, 1973: Axelson M, Sjovall J, Gustafsson BE, Setchell KD. Soya a dietary source of the nonsteroidal oestrogen equol in man and animals. J Endocrinol 1984;102: Thompson LU, Robb P, Serraino M, Cheung F. Mammalian lignan production from various foods. Nutr Cancer 1991;16: Price KR, Fenwick GR. Naturally occurring oestrogens in foods a review. Food Addit Contam 1985;2: Knight DC, Eden JA. A review of the clinical effects of phytoestrogens. Obstet Gynecol 1996;87: Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 1991;20: Stampfer MJ, Colditz GA, Willett WC, et al. Postmenopausal estrogen therapy and cardiovascular disease. N Engl J Med 1991;325: Adlercreutz H, Markkanen H, Watanabe S. Plasma concentrations of phyto-oestrogens in Japanese men. Lancet 1993;342: Hertog MG, Kromhout D, Aravanis C, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med 1995;155: Adlercreutz H, Hamalainen E, Gorbach S, Goldin B. Dietary phytooestrogens and the menopause in Japan. Lancet 1992;339: Miksicek RJ. Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol 1993;44: Honore EK, Williams JK, Anthony MS. Enhancement of coronary vasodilation by soy phytoestrogens and genistein [abstract]. Circulation 1995;92:I Jiang C, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Endothelium-independent relaxation of rabbit coronary artery by 17beta-oestradiol in vitro. Br J Pharmacol 1991;104: Sudhir K, Chou TM, Mullen WL, et al. Mechanisms of estrogeninduced vasodilation: in vivo studies in canine coronary conductance and resistance arteries. J Am Coll Cardiol 1995;26: Chester AH, Jiang C, Borland JA, Yacoub MH, Collins P. Estrogen relaxes human epicardial coronary arteries through non-endotheliumdependent mechanisms. Coronary Artery Dis 1995;6: Collins P, Rosano GMC, Jiang C, Lindsay D, Sarrel PM, Poole- Wilson PA. Hypothesis: cardiovascular protection by estrogen a calcium antagonist effect? Lancet 1993;341: Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990;101: Ferreira SH, Moncada S, Vane JR. Further experiments to establish that the analgesic of aspirin-like drugs depends on the inhibition of prostaglandin biosynthesis. Br J Pharmacol 1973;47:629P 30P. 24. Quayle JM, Standen NB, Stanfield PR. The voltage-dependent block of ATP-sensitive potassium channels of frog skeletal muscle by caesium and barium ions. J Physiol 1988;405: Wakeling AE, Dukes M, Bowler J. A potent specific pure antiestrogen with clinical potential. Cancer Res 1991;51: Martin W, Villani GM, Jothianandan D, Furchgott RF. Blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation of rabbit aorta by certain ferrous hemoproteins. J Pharmacol Exp Ther 1985;233: Terracciano CM, MacLeod KT. Effects of acidosis on Na /Ca2 exchange and consequences for relaxation in guinea pig cardiac myocytes. Am J Physiol 1994;267:H Jiang C, Poole-Wilson PA, Sarrel PM, Mochizuki S, Collins P, MacLeod KT. Effect of 17beta-oestradiol on contraction, Ca 2 current and intracellular free Ca 2 in guinea-pig isolated cardiac myocytes. Br J Pharmacol 1992;106: Jiang C, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Progesterone induces endothelium-independent relaxation of rabbit coronary artery in vitro. Eur J Pharmacol 1992;211: Opie LH, editor. Drugs for the Heart. 4th ed. Philadelphia Saunders, McDonald TF, Pelzer S, Trautwein W, Pelzer DJ. Regulation and modulation of calcium channels in cardiac, skeletal and smooth muscle cells. Physiol Rev 1994;74: Yang SG, Saifeddine M, Hollenberg MD. Tyrosine kinase inhibitors

9 JACC Vol. 35, No. 7, 2000 June 2000: Figtree et al. Plant Estrogens and Coronary Arteries 1985 and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle. Can J Physiol Pharmacol 1992;70: Jin N, Siddiqui RA, English D, Rhoades RA. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am J Physiol 1996;271:H Steusloff A, Paul E, Semenchuk LA, Di-Salvo J, Pfitzer G. Modulation of Ca2 sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation. Arch Biochem Biophys 1995; 320: Tsuda T, Kawahara Y, Shii K, Koide M, Ishida Y, Yokoyama M. Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells. FEBS Lett 1991;285: Abebe W, Agrawal DK. Role of tyrosine kinases in norepinephrineinduced contraction of vascular smooth muscle. J Cardiovasc Pharmacol 1995;26: Di Salvo J, Steusloff A, Semenchuk L, Satoh S, Kolquist K, Pfitzer G. Tyrosine kinase inhibitors suppress agonist-induced contraction in smooth muscle. Biochem Biophys Res Commun 1993;190: Horwitz KB, Horwitz LD. Canine vascular tissues are targets for androgens, estrogens, progestins and glucocorticoids. J Clin Invest 1982;69: Ingegno MD, Money SR, Thelmo W, et al. Progesterone receptors in the human heart and great vessels. Lab Invest 1988;59: Wang TT, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis 1996;17: Xu X, Wang HJ, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr 1994;124: Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93: Gustafsson JA. Estrogen receptor beta Getting in on the action? Nature Med 1997;3: Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995;268: C Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, Nelson MT. Hyperpolarizing vasodilators activate ATP-sensitive K channels in arterial smooth muscle. Science 1989;245: Wagner JD, Martino MA, Jayo MJ, Anthony MS, Clarkson TB, Cefalu WT. The effects of hormone replacement therapy on carbohydrate metabolism and cardiovascular risk factors in surgically postmenopausal cynomolgus monkeys. Metabolism 1996;45: Balmir F, Staack R, Jeffrey E, Jimenez MD, Wang L, Potter SM. An extract of soy flour influences serum cholesterol and thyroid hormones in rats and hamsters. J Nutr 1996;126: Stephan EB, Dziak R. Effects of genistein, tyrphostin and pertussis toxin on EGF-induced mitogenesis in primary culture and clonal osteoblastic cells. Calcif Tissue Int 1994;54: Anderson JB, Garner SC. The effects of phytoestrogens on bone. Nutr Res 1997;17: Cline JM, Paschold JC, Anthony MS, Obasanjo IO, Adams MR. Effects of hormonal therapies and dietary soy phytoestrogens on vaginal cytology in surgically postmenopausal macaques. Fertil Steril 1996;65: Anthony MS, Clarkson TB, Hughes CL, Jr, Morgan TM, Burke GL. Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. J Nutr 1996;126:43 50.