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Endocrinology, doi:10.1210/en.2007-1361
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Endocrinology Vol. 149, No. 5 2678-2687
Copyright © 2008 by The Endocrine Society

Intracoronary Genistein Acutely Increases Coronary Blood Flow in Anesthetized Pigs through β-Adrenergic Mediated Nitric Oxide Release and Estrogenic Receptors

Elena Grossini, Claudio Molinari, David A. S. G. Mary, Francesca Uberti, Philippe Primo Caimmi, Nicola Surico and Giovanni Vacca

Laboratorio di Fisiologia, Dipartimento di Medicina Clinica e Sperimentale, Facoltà di Medicina e Chirurgia, Università del Piemonte Orientale "A. Avogadro," I-28100 Novara, Italy

Address all correspondence and requests for reprints to: Dott. Elena Grossini, Facoltà di Medicina e Chirurgia, via Solaroli 17, I-28100 Novara, Italy. E-mail: grossini{at}med.unipmn.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Statistical analyses
 Results
 Discussion
 References
 
Various studies have suggested that the phytoestrogen genistein has beneficial cardioprotective and vascular effects. However, there has been scarce information regarding the primary effect of genistein on coronary blood flow and its mechanisms including estrogen receptors, autonomic nervous system, and nitric oxide (NO). The present study was planned to determine the primary effect of genistein on coronary blood flow and the mechanisms involved. In anesthetized pigs, changes in left anterior descending coronary artery caused by intracoronary infusion of genistein at constant heart rate and arterial pressure were assessed using ultrasound flowmeters. In 25 pigs, genistein infused at 0.075 mg/min increased coronary blood flow by about 16.3%. This response was graded in a further five pigs by increasing the infused dose of the genistein between 0.007 and 0.147 mg/min. In the 25 pigs, blockade of cholinergic receptors (iv atropine; five pigs) and {alpha}-adrenergic receptors (iv phentolamine; five pigs) did not abolish the coronary response to genistein, whose effects were prevented by blockade of β2-adrenergic receptors (iv butoxamine; five pigs), nitric oxide synthase (intracoronary N{omega}-nitro-L-arginine methyl ester; five pigs) and estrogenic receptors (ERs; ER{alpha}/ERβ; intracoronary fulvestrant; five pigs). In porcine aortic endothelial cells, genistein induced the phosphorylation of endothelial nitric oxide synthase and NO production through ERK 1/2, Akt, and p38 MAPK pathways, which was prevented by the concomitant treatment by butoxamine and fulvestrant. In conclusion, genistein primarily caused coronary vasodilation the mechanism of which involved ER{alpha}/ERβ and the release of NO through vasodilatory β2-adrenoreceptor effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Statistical analyses
 Results
 Discussion
 References
 
THERE HAS BEEN extensive research regarding the cardiovascular benefits of estrogen and its side effects, leading to the exploration of alternative estrogen receptor modulators, particularly in the form of the phytoestrogen genistein. For instance, in women coronary heart disease has been linked to loss of estrogen protection (1, 2), and estrogen administration is associated with reduced cardiovascular risk factors (3, 4, 5). In animal models the administration of 17β-estradiol has been shown to cause vasodilation through the release of nitric oxide (NO) (6). However, estrogen therapy has been linked to increased incidence of complications including malignancy (7, 8) and venous thrombosis (9). Alternative substances related to estrogen have been examined, of which the naturally occurring phytoestrogens with flavonoid structure have gained a wide attention. Epidemiological data have indicated that consumption of diets rich in such phytoestrogens was associated with a low incidence and mortality of coronary artery disease (10, 11, 12). Also, soy isoflavones have been reported to have favorable effect on lipids (11), vascular reactivity (13), and formation of atherosclerotic lesions (14).

The effect of the phytoestrogen genistein on the vasculature has been previously examined. It has been reported to result in vasodilation of vascular beds in animals (15, 16, 17, 18) and humans (19, 20, 21). The mechanism of this effect has been variably attributed to endothelial (13, 15, 16, 18, 19, 20, 21) and nonendothelial factors (21, 22, 23). In respect of the coronary circulation, genistein has been reported to have a protective effect against ischemic-reperfusion injury (24, 25, 26), which involved the subtype-β of the estrogen receptors (ER; ERβ) (26). Also, genistein was found to cause vasodilation in coronary artery preparations of animal (22, 23, 27, 28, 29, 30), although the mechanisms involved were either attributed to endothelial (22, 27) or other unrelated factors (23, 28, 29, 30). Finally, genistein has been shown to have an inotropic effect in canine ventricular trabeculae (31), which can secondarily affect coronary blood flow. The above considerations indicate that the primary effect of genistein on the intact coronary circulation, and its mechanisms are as yet not unequivocally demonstrated, although a vasodilation related to endothelial function and Erβ remains as a hypothetical possibility.

The present work was therefore planned, in controlled experiments in anesthetized pigs, to investigate the primary in vivo effect of the administration of genistein on coronary blood flow and determine the mechanisms involved. This was achieved by infusing the isoflavone locally into the coronary circulation, whereas preventing changes in heart rate and arterial blood pressure to avoid the secondary interference by reflex and local metabolic and physical effects. In addition, the intracellular pathway related to NO mechanisms involved in the vascular effects elicited by genistein was examined in porcine aortic endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Statistical analyses
 Results
 Discussion
 References
 
In vivo study
The experiments were carried out in prepubertal female 30 pigs, weighing 65–71 kg, supplied by an accredited dealer (Azienda Invernizzi, Olengo, Novara, Italy). The animals were fasted overnight and then anesthetized with im ketamine (20 mg/kg; Parke-Davis, Detroit, MI) followed after about 15 min by iv sodium pentobarbitone (15 mg/kg; Siegfried, Zofingen, Germany), after which they were artificially ventilated with oxygen-enriched air using a respiratory pump (Harvard 613; Harvard Apparatus, South Natick, MA). Anesthesia was maintained throughout the experiments by continuous iv infusion of sodium pentobarbitone (7 mg/kg·h) and assessed as previously reported (32) from responses of the animals to somatic stimuli. The experiments were carried out in accordance with national guidelines (D.L.G.S. 27/01/1992, license no. 116). This study was monitored in accordance with Good Research Practice Guidelines in the European Community and the Guide for the Care and Use of Laboratory Animals [1996 (7th ed.) Washington, DC: National Academy Press, National Research Council Guide].

Pressures in the ascending aorta and the right atrium were recorded via catheters connected to pressure transducers (Statham P23 XL; Gould, Valley View, OH) inserted into the right femoral artery and the right external jugular vein, respectively. The chest was opened in the left fourth intercostal space, the pericardium was cut, and an ultrasound flowmeter probe (model 420; Transonic Systems Inc., Ithaca, NY) was positioned around the proximal part of the anterior descending coronary artery to record coronary blood flow. Left ventricular pressure was measured by means of a catheter connected to a pressure transducer (Gould) inserted through the left atrium. The frequency response of the catheter-manometer system was found to be flat (± 5%) up to 40 Hz. To pace the heart, electrodes were sewn on the left atrial appendage and connected to a stimulator (model S8800; Grass Instruments, Quincy, MA), which delivered pulses of 3–5 V for 2 msec duration at the required frequency. Arterial blood samples were used to measure pH and arterial pressure of oxygen and carbon dioxide (PO2 and PCO2) (with a gas analyzer; ABL 505; Radiometer, Copenhagen, Denmark) and the hematocrit. The acid-base status of the animals was kept within normal limits as previously reported (32). Genistein (Sigma, Milan, Italy) was infused in the left anterior descending coronary artery by using a catheter connected to a butterfly needle inserted into the artery distal to the flowmeter probe.

To prevent changes in aortic pressure during the experiments, a large-bore cannula was introduced into the abdominal aorta through the left femoral artery and connected to a reservoir containing Ringer’s solution and kept at 38 C. The reservoir was pressurized using compressed air, which was controlled with a Starling resistance, and pressure within the reservoir was measured by a mercury manometer. This procedure has been shown to allow the aortic pressure to be maintained at steady levels without significant changes in filling pressures of the heart or the hematocrit (6, 33). Coagulation of the blood was avoided by the iv injection of heparin (Parke Davis; initial doses of 500 IU/kg and subsequent doses of 50 IU/kg every 30 min). The rectal temperature of the pigs was monitored and kept between 38 C and 40 C using an electric pad.

Mean and phasic aortic blood pressure, mean right atrial pressure, left ventricular pressure, mean and phasic coronary blood flow, heart rate, and the maximum rate of change of left ventricular systolic pressure (dP/dtmax), were recorded via a micro1401 A/D converter [Cambridge Electronic Design (CED), Cambridge, UK] and processed by using Spike2 Software (CED). The heart rate was obtained from the electrocardiogram. The frequency response of the differentiator used to obtain left ventricular dP/dtmax was flat (±5%) up to 150 Hz.

To calculate coronary vascular resistance, the difference between mean aortic blood pressure and mean left ventricular pressure during diastole was considered as the coronary pressure gradient. Coronary vascular resistance was calculated as the ratio between this pressure gradient and mean diastolic coronary blood flow. The diastolic period of measurement was defined as starting when ventricular pressure reached its minimum value after systole and ended when it increased at the end of diastole.

At the end of the experiment, each animal was killed by an iv injection of 90 mg/kg sodium pentobarbitone.

In vitro study
Porcine aortic endothelial (PAE) cells were cultured in DMEM (Sigma) supplemented with 10% fetal calf newborn serum (Sigma) and 1% penicillin-streptomycin-glutamine (Sigma). Cultured cells were maintained at 37 C with 5% CO2. In the first set of experiments, 1 x 104 cells were plated in 96-well plates with DMEM 0% fetal calf newborn serum supplemented with 1% penicillin-streptomycin-glutamine without red phenol (starvation medium) for 4 or 18 h; in the second set of experiments, the cells were plated on the dishes with starvation medium for 18 h.

Experimental protocol
In vivo study.
The experiments were begun after at least 30 min of steady-state conditions with respect to measured hemodynamic variables. In the 30 pigs, the heart was paced to a frequency higher, by 20 beats/min, than that observed during the steady state, and the arterial system was connected to the pressurized reservoir. After at least 10 min of steady-state conditions, the experiments were carried out by intracoronary infusing, in each pig, either a solution of genistein obtained by dissolving the substance in saline containing 2.5% dymethilsulfoxide (DMSO) or the vehicle only. The infusions were completed within a period of 5 min by using an infusion pump (model 22; Harvard Apparatus) working at constant rate of 1 ml/min. In 25 pigs, a dose of 0.075 mg/min of genistein (0.001 mg/min for each milliliter per minute of measured coronary blood flow; 3.7 µM) was infused into the coronary artery. This dose corresponded to the reported dose of 0.5 mg/kg of body weight given iv as bolus administration (24), considering the mean value of cardiac output (not shown) and coronary blood flow. After the infusion was stopped, observations were continued for 15 min.

Recordings taken for 10 min during the steady-state before infusion of genistein were used as control. Measurements of hemodynamic variables were obtained during the last 30 sec of infusion in the steady state and compared with control values. The effect of infusion of genistein on coronary blood flow and the mechanisms involved were studied in 25 pigs. In the remaining five pigs, the effects of graded administration of genistein were examined by infusing the substance at eight subsequent doses of 0.007, 0.015, 0.029, 0.059, 0.074, 0.088, 0.118, and 0.147 mg/min, which corresponded to doses of genistein ranging from 0.05 to 1 mg/kg. Each dose was calculated as described above and infused for 5 min. The resulting changes in coronary blood flow were compared with control values obtained before starting the infusion.

The mechanisms of the response of coronary blood flow to the infusion of genistein were studied in the group of 25 pigs by repeating the experiment after hemodynamic variables had returned to control levels. In five pigs genistein was administered after the iv administration of atropine sulfate (0.5 mg/kg; Sigma); five pigs after the iv administration of phentolamine (1 mg/kg; Ciba Geigy, Basel, Switzerland); five pigs after the iv administration of butoxamine (2.5 mg/kg; Sigma); five pigs after the intracoronary administration of N{omega}-nitro-L-arginine methyl ester (L-NAME; 2 mg for each milliliter per minute of coronary blood flow; Sigma); and five pigs after the intracoronary administration of 0.14 ± 0.04 (0.097–0.21) mg/min of fulvestrant (Sigma). All the drugs were given in the absence of pacing of the heart and without controlling aortic pressure; their effects on hemodynamic variables were measured in the steady state. In all subsequent experiments, changes in heart rate and aortic pressure were prevented.

In vitro study. No assay.
In the first set of experiments NO production from PAE cells was measured in culture supernatants using the Griess method (Promega, Madison, WI), following the manufacturer’s instruction. Cells plated in 96-well plates were maintained in starvation medium for 18 h and then treated with the β-adrenergic nonspecific (10 µM isoproterenol; Sigma) and specific (10 µM salbutamol; Sigma) agonists, the β2-adrenergic-specific antagonist butoxamine (100 µM; Sigma), 17β-estradiol (100 nM; Sigma), the adenyl cyclase activator forskolin (1 µM, Sigma), the adenyl cyclase blocker 2'-5'dideoxyadenosine (1 µM; Sigma) dissolved in DMSO, L-NAME (10 mM; Sigma), and acetylcholine (10 µM; Sigma) in the presence or absence of genistein (1 µM-10 µM) dissolved in DMSO. In some experiments, PAE cells were pretreated with the estrogen receptor inhibitor fulvestrant (10 µM; Sigma), p38 MAPK inhibitor SB203580 (1 µM; Promega), phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (100 nM; Sigma), or MAPK kinase-1 inhibitor UO126 (10 µM; Promega) given 15 min before the addition of other agents used before. These inhibitors and their vehicle were tested in the basal medium without agents as well. At the end of stimulation, the sample’s supernatants were mixed with equal volume of Griess reagents and after 10 min the absorbance at 570 nm was measured by a spectrometer (BS1000 Spectra Count, San Jose, CA). The NO production was calculated as previously described in detail (34). The results obtained with the Griess method have been validated also through the diaminofluorescein (DAF-FM) fluorophore system (Molecular Probes, Invitrogen, Carlsbad, CA) used as previously described (35). Briefly the cells were plated in 96-well plates and maintained in starvation medium for 4 h before the treatment with the same agents and conditions used for Griess assay. At the end of stimulations, the cells were washed with PBS 1x sterile and incubated with PBS 1x sterile with DAF-FM diacetate (0.8 µM) for 15 min in the dark in incubator at 37 C with 5% CO2. The cell-bathing medium was taken for measurement of fluorescence by fluorescence spectometer. Fluorescence excitation and emission were 495 and 515 nm, respectively. To determine the range of reliable fluorescence readouts, a standard curve for DAF-FM diacetate fluorescence was generated using detanonoate (1, 5, 10, 50, 100 200, 400 µM; Sigma) (35) incubated with DAF-FM diacetate (0.8 µM) for 20 min in the dark in incubator at 37 C with 5% CO2. The fluorescence of samples was measured by a fluorescence spectometer at {lambda}ex/em 495/515 nm.

Western blot analysis.
To characterize the effects of genistein, in the second set of experiments, PAE cells were cultured in the dishes with starvation medium for 18 h. The cells were incubated and pretreated with the same agents and inhibitors at the same concentration used for Griess assay. Stimulations were blocked by washing with 1x iced PBS supplemented with 1:200 sodium orthovanadate (Sigma) and addition of ice-lysis buffer (HEPES 25 mM, NaCl 135 mM, Nonidet P-40 1%, EDTA 5 mM, EGTA 5 mM, ZnCl2 1 mM, NaF 50 mM, glycerol 10%) supplemented with 1:200 sodium orthovanadate and 1:100 protease-inhibitor cocktail (Sigma). The cells were solubilized for 15 min at 4 C. The extract proteins were quantified by BCA assay (Pierce, Rockford, IL), and the same quantity of protein was dissolved in Laemmli buffer 5 times, heated for 5 min at 95 C, and resolved on 10% SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to polyvinyl difluoride membranes (Bio-Rad), which were incubated overnight with specific primary antibodies: anti phospho-ERK (Thr202/Tyr204; Cell Signaling, Beverly, MA), anti ERK1/2, antiphospho-Akt (Ser473; Cell Signaling), anti Akt, antiphospho-p38 (Thr180/Tyr182; Cell Signaling), anti-p38, antiphospho-endothelial NO synthase (eNOS; Ser1179; Alexis Biochemicals, Lausen, Switzerland), and anti-eNOS. The immunoreactive proteins were visualized with horseradish peroxidase-coupled goat-antirabbit IgG and goat antimouse IgG using Immun-star horseradish peroxidase substrate kit (Bio-Rad). Phosphorylated protein expression was normalized through specific total protein expression and verified through β-actin detection.


   Statistical analyses
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
Results
 Discussion
 References
 
Student’s paired t test was used to examine changes in measured variables caused by genistein infusion. ANOVA for repeated measurements was used to examine the effects of successive procedures on coronary blood flows. Multiple comparisons between data were made by the Newman-Keuls test. A value of P < 0.05 was considered statistically significant. Considering the in vivo experiments, group data (obtained as described in Experimental protocol) are presented as mean ± SD (range). Regarding the in vitro experiments, all results, presented as mean ± SD (range), were obtained from five different cell cultures for each experimental protocol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
 Results
Discussion
 References
 
In vivo study
In all pigs, recordings commenced approximately 5 h after the induction of anesthesia. The mean pH, PO2, and PCO2 of arterial blood were 7.40 ± 0.03 (7.39–7.43), 118.3 ± 9.3 (102–142) mm Hg and 39.7 ± 1.1 (37–42) mm Hg and the hematocrit was 38.5% ± 1.6 (38–40).

Responses to intracoronary infusion of genistein
In the 25 pigs, intracoronary infusion of the vehicle did not cause changes in the control values of hemodynamic variables. Group values of data and individual changes in hemodynamic parameters caused by intracoronary infusion of genistein, and preventing changes in heart rate and aortic blood pressure, are shown in Table 1Go and Fig. 1BGo, respectively.


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  		TABLE 1. Changes in hemodynamic variables caused by intracoronary infusion of 0.075 mg/min of genistein in 25 pigs

 

Figure 1
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  		FIG. 1. Hemodynamic effects of intracoronary infusion of genistein in anesthetized pigs. A, Example of experimental recordings showing the hemodynamic effects of the intracoronary infusion of genistein in one pig. From top to bottom: heart rate (HR), mean and phasic aortic blood pressure (ABP), left ventricular pressure (LVP), dP/dtmax, mean right atrial pressure (RAP), and mean and phasic coronary blood flow (CBF). The arrows indicate the beginning and end of the infusion. B, Response of CBF to the intracoronary infusion of genistein in 25 pigs. The values of CBF obtained during the test period of measurement are plotted on the ordinate against the corresponding values before infusion on the abscissa. The continuous line is the line of equality. C, Response of CBF to graded increases in the infused dose of genistein from 0.007 and 0.147 mg/min in five pigs. The means of percentage changes in blood flow obtained in the five pigs during the test period of measurement are plotted against the logarithm of the doses. C, Control value before infusions. The bars indicate SD.

 
In each pig, intracoronary infusion of genistein caused an increase in mean coronary blood flow by 10.1 ± 3.6 (4.9–20.4; P < 0.05) ml/min, corresponding to a decrease in coronary vascular resistance of 0.27 ± 0.06 (0.19–0.46; P < 0.05) mm Hg/ml·min. The increase of coronary blood flow amounted to about 16.3 ± 4.3% (11.3–26.8%), whereas the decrease of coronary vascular resistance amounted to about 25.5 ± 4.3% (18.4–35.9%) of the control values.

The coronary effects of genistein began within about 90 sec after starting the infusion, reached a steady state in about 2 min and were over within 5 min after the end of the infusion. Changes of dP/dtmax, right atrial pressure and left ventricular pressure were not significant (Table 1Go).

An example of experimental recording taken in one of the 25 pigs is shown in Fig. 1AGo.

Dose-response study
In the five pigs, the intracoronary infusion of the eight doses of genistein caused a dose-response effect on coronary blood flow as illustrated in Fig. 1CGo. The control value of mean coronary blood flow was 61.8 ± 6.5 (53–71) ml/min. The threshold dose of the hormone was found at a dose of 0.015 mg/min. Maximal effect was observed at a dose of 0.118 mg/min.

Mechanisms of the responses
As reported in Fig. 2Go, A and B, in 10 pigs, blockade of cholinergic and {alpha}-adrenergic receptors did not affect the coronary responses to the intracoronary infusion of genistein. After giving atropine (five pigs) and phentolamine (five pigs), group increases of coronary blood flow amounted, respectively, to 10.7 ± 3.5 (5.2–15; P< 0.05) ml/min and 9.5 ± 3.6 (5–14.3; P < 0.05) ml/min from control values. In the same pigs, the increase of coronary blood flow caused by intracoronary genistein before giving atropine and phentolamine was, respectively, 10.9 ± 3.1 (5.7–13.8; P<0.05) ml/min and 9.4 ± 3.5 (4.9–14.2; P < 0.05) ml/min from control values. The difference between the responses before and after giving the blocking agents was not significant (Fig. 2Go, A and B; P > 0.05).


Figure 2
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  		FIG. 2. Coronary blood flow (CBF) effects induced by the intracoronary infusion of genistein before and after blockade of muscarinic cholinoceptors (A; n = 5), {alpha}-adrenoceptors (B; n = 5), β2-adrenoceptors (C; n = 5), the NO synthase (D; n = 5), and estrogenic receptors (E; n = 5). Genistein was infused before (filled columns) and after (open columns) giving atropine (A), phentolamine (B), butoxamine (C), L-NAME (D), and fulvestrant (E).

 
As shown in Fig. 2Go, C–E, the blockade of β2-adrenergic receptors, the eNOS and the ER{alpha}/ERβ abolished the coronary effects of intracoronary infusion of genistein.

After giving butoxamine (five pigs), the increase of coronary blood flow amounted to 0.3 ± 0.4 (0–1; P > 0.05) ml/min from control values (Fig. 2CGo). In the same pigs, before giving butoxamine, intracoronary genistein administration caused an increase of coronary blood flow of 9.3 ± 2.3 (7.3–12.2; P < 0.05) ml/min from control values.

After giving L-NAME (five pigs) and fulvestrant (five pigs), the administration of genistein increased coronary blood flow, respectively, by 0.2 ± 0.4 (0–1; P > 0.05) ml/min and 0.4 ± 0.5 (0–1; P > 0.05) ml/min from control values (Fig. 2Go, D and E). In the same pigs, before giving the blocking agents, the coronary blood flow effects amounted, respectively, to 14.3 ± 8 (6.6–25; P < 0.05) ml/min and 10 ± 4.1 (5.2–15.2; P < 0.05) ml/min of control values.

In vitro study
Genistein acutely induces NO release.
The effects of the isoflavone on NO release were examined in PAE cells, cultured as described in Materials and Methods. Concentrations of genistein ranging from 1 to 10 µM were used and tested for 1, 2, and 10 min. The optimal time course chosen was 1 min because after 2 min the effects on NO production progressively decreased (Fig. 3AGo). These results on NO production were not different from those obtained with the DAF-FM system (Fig. 3BGo). The effect of genistein was concentration dependent, when genistein 10 µM induced the maximal NO production in PAE cells (93.75% of control values; Fig. 3AGo and Table 2Go). This concentration of genistein was used for all successive experiments. The effects of genistein were abolished in the presence of L-NAME (10 mM, Fig. 3CGo).


Figure 3
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  		FIG. 3. Effects of genistein on NO production in PAE cells. A, Means of percent increases in NO production obtained during the test period of measurement are plotted against the doses of genistein (G; 1–10 µM) administered for 1, 2, or 10 min. These percent values correspond to the NO (micromoles) produced by samples containing 1.5 µg of proteins each. B, NO production measurement (percent) by DAF-FM diacetate fluorescence method induced by genistein (1, 5, 10 µM) for 1, 2, or 10 min The calibration curve for DAF-FM was obtained through detanonoate (1, 5, 10 µM). C, NO production (percent) in PAE cells induced by genistein in presence of various agents is reported (1 min stimulation). G, Genistein; Is, isoproterenol; Sal, salbutamol; Is + G, costimulation; Sal+G, costimulation; N+G, costimulation L-NAME + G; N+ Is + G, costimulation in presence of L-NAME; N+ Sal + G, costimulation in presence of L-NAME; B, butoxamine; B+G, costimulation; E, 17 β estradiol; Ach, acetylcholine. D, NO production measurement (percent) in PAE cells caused by various agents in presence of fulvestrant (Ic) is reported. E, NO production measurement (percent) in PAE cells caused by various agents in presence of wortmannin, UO126, and SB203580 (In) is reported. F, NO production measurement (percent) in PAE cells caused by various agents in presence of 2'-5'dideoxyadenosine (2'-5'D) is reported. F, Forskolin. All results were replicated in five independent experiments performed in five different cell cultures and are reported as means ± SD (indicated by the bars). *, Effect of DMSO. a, b, c, h, i, and z, P < 0.05 vs. control; d, g, j, p, and u, P < 0.05 vs. a; k, q, and v, P < 0.05 vs. b; l, r, and w, P < 0.05 vs. c; e, m, s, and x, P < 0.05 vs. Is+ G; f, n, t, and y, P < 0.05 vs. Sal+G; o, P < 0.05 vs. f.

 

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  		TABLE 2. Changes in level of NO production caused by various agents

 
Involvement of β2-adrenergic receptor in the induction of NO release by genistein.
To examine whether genistein could interact with β2-adrenergic signaling to activate NO release, experiments were performed in the presence of β-adrenergic agonists and antagonists. Administration of the 10 µM β-adrenergic nonspecific agonist isoproterenol or 10 µM β2-adrenergic-specific agonist salbutamol caused a significant increase of NO production (86.25 and 90% of control values, respectively), an effect that was amplified by genistein (141.8 and 166.7% of control values, respectively; Fig. 3Go, C–F, and Table 2Go), confirming previously reported findings (17) and the hypothesis of interaction between genistein and β-adrenergic receptor. To further confirm these findings, PAE cells were treated with butoxamine in costimulation with genistein. As shown in Fig. 3CGo, butoxamine, having itself caused a reduction of NO release of about 5%, reduced the effects of genistein by about 95%. Furthermore L-NAME abolished the effects of genistein given in costimulation with either isoproterenol or salbutamol (Fig 3CGo). Taken together, these findings confirm the involvement of genistein in β2-adrenergic signaling leading to NO synthesis.

Role of cAMP in the intracellular signaling leading to NO production.
Because the activation of NO synthesis by genistein involved the β2-adrenergic signaling and because of reports showing a role of cAMP in effects induced by both genistein and β-agonists leading to NO production (36, 37), experiments were performed to confirm those reports in PAE cells as well. The PAE cells were pretreated with the specific inhibitor of adenyl cyclase (2'-5'dideoxyadenosine, 1 µM) for 30 min and then stimulated with 10 µM genistein and isoproterenol or salbutamol alone or together. This concentration of 2'-5'dideoxyadenosine was found to prevent the effects of forskolin 1 µM on NO production (Table 2Go). As reported in Fig. 3FGo and Table 2Go, the effects of genistein, given alone or in costimulation with isoproterenol or salbutamol, were abolished, providing evidence for a role of cAMP in the response of PAE cells to the phytoestrogen and to the β-agonists leading to NO.

β2-Adrenergic-mediated NO production caused by genistein is modulated by ER{alpha}/ERβ.
Because genistein has been reported to elicit beneficial cardiovascular effects through estrogenic receptors, experiments were performed in PAE cells pretreated for 15 min with 10 µM fulvestrant and then stimulated with 10 µM genistein, 10 µM isoproterenol, or salbutamol alone or in costimulation. The dose of fulvestrant was found to prevent the NO production caused by 17β-estradiol (Fig. 3DGo) and is similar to the one previously reported (38) to prevent the effects of equimolar doses of genistein. In the samples pretreated with fulvestrant, genistein, and isoproterenol or salbutamol alone caused no significant changes in NO levels in respect of control values, whereas the effects of the costimulation of genistein and isoproterenol or salbutamol were, respectively, reduced of about 88.5% and abolished (Fig. 3DGo and Table 2Go). These findings show that the actions of genistein involved activation of ER{alpha}/ERβ, thus confirming the results obtained in the anesthetized pigs. Interestingly, in the presence of fulvestrant, neither isoproterenol nor salbutamol caused any change in NO release thus supporting the presence of an interrelation between β2-adrenergic receptors and estrogenic receptors pathways leading to NO production.

Increased NO production by genistein is regulated by PI3K, MAPK, and p38/MAPK.
To elucidate the intracellular signaling involved in the activation of NO production, the role of PI3K/Akt, ERK/MAPKs, and p38/MAPK pathways was examined. Preincubation for 15 min of PAE cells with 100 nM wortmannin, 10 µM UO126, and 1 µM SB203580 abolished the effects on NO production induced by 10 µM genistein and 10 µM isoproterenol or salbutamol alone and in costimulation (Fig. 3EGo and Table 2Go). These findings confirm previously reported data (34) obtained in PAE cells about the role of PI3K/Akt, ERK/MAPKs, and p38/MAPK in the signaling of β2-adrenoceptors and show that genistein and β-agonists share the same intracellular pathways to activate NO synthesis.

Analysis of intracellular pathway leading to eNOS phosphorylation.
Because the results obtained with the Griess assay show that genistein stimulates NO production, the effects on eNOS activity were examined by analyzing the level of phosphorylation induced by the agents used in previous experiments (genistein, isoproterenol, salbutamol, butoxamine, fulvestrant given alone or in costimulation). 17β-Estradiol was tested as positive control because estrogens have been shown to rapidly activate eNOS through a signaling pathway initiated from membrane ER (39). Immunoblots and the densitometric analysis showed that in presence of genistein the level of eNOS phosphorylation was greater than control. Moreover genistein potentiated the effect of both isoproterenol and salbutamol. In presence of L-NAME and fulvestrant, the effects of genistein, given alone, and isoproterenol and salbutamol, given in costimulation with genistein, were abolished. Furthermore, butoxamine almost prevented any effect on the level of phosphorylation induced by genistein (Fig. 4AGo).


Figure 4
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  		FIG. 4. The effect of various agents on the level of phosphorylation (percent) of eNOS (A), Akt (B), ERK 1/2 (C), and p38 (D). In each panel both the densitometric analysis and immunoblots of phosphorylation relative to specific proteins are represented. These experiments were performed in five different cell cultures obtained from five different porcine aortae. The effect of various agents on the level of phosphorylation in the absence or presence of Ic and/or In is reported. Results are reported as means ± SD (indicated by the bars). The abbreviations are the same used in Fig. 3Go. N, L-NAME; D, DMSO. a, b, c, and p, P < 0.05 vs. control; f, i, j, and m, P < 0.05 vs. a; d, P < 0.05 vs. b; e, P < 0.05 vs. c; g, k, and n, P < 0.05 vs. d; h, l, and o, P < 0.05 vs. e; q, P < 0.05 vs. p.

 
To confirm that eNOS activation was mediated by PI3K/Akt, ERK/MAPKs, and p38/MAPK pathways, experiments were repeated before and after administration of 100 nM wortmannin, 10 µM UO126, and 1 µM SB203580. 17β-Estradiol was tested as positive control again. As shown in Fig. 4Go, B–D, in absence of inhibitors, genistein and isoproterenol or salbutamol given alone or in costimulation caused an increase of phosphorylation of ERK1/2, Akt, and p38; when these agents were administered in presence of L-NAME and fulvestrant, the levels of phosphorylation were similar to the basal samples (Fig. 4Go, B–D).

Butoxamine abolished the effect of genistein on the levels of phosphorylation of ERK1/2, Akt, and p38. Finally, in presence of the inhibitors, any changes in the levels of phosphorylation of ERK1/2, Akt, and p38 induced by the various agents were totally abolished (Fig. 4Go, B–D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analyses
 Results
 Discussion
References
 
The present study demonstrates for the first time in the anesthetized pig that the local administration of genistein into the coronary circulation increased coronary blood flow in the absence of changes of heart rate, arterial and cardiac filling pressures, and myocardial contractility. The mechanisms of this response involved ER{alpha}/ERβ and an increase of NO release through vasodilatory β2-adrenoreceptor effects.

In vivo studies
In the present study, the primary effect of genistein on the coronary circulation was examined by performing the experiments in vivo and preventing changes in heart rate and arterial pressure and in the absence of changes in cardiac filling pressures and myocardial contractility. In addition, genistein was locally administered into the coronary circulation using doses similar to those previously used (24) after adjusting for the measured coronary blood flow and cardiac output. Thus, the design of the experiments helped avoid any secondary interference from reflex and physical effects on the observed responses of the coronary circulation and the related cardiac function (40). Furthermore, none of these responses to genistein could be obtained during intracoronary infusion of the vehicle alone at the same rate as that of genistein. Finally, the direct relationship between the phytoestrogen and its coronary response was confirmed during the dose-response study, which showed that the increase in coronary blood flow could be augmented by increasing the dose of the infused substance. Therefore, the present investigation showed that intracoronary infusion of genistein primarily caused coronary vasodilation because this response did not involve changes in other hemodynamic variables.

The demonstration of the above primary effects of genistein is consistent with previously reported findings obtained through in vitro and in vivo studies. For instance, genistein has been reported to cause vasodilation of isolated porcine coronary arteries (29), porcine (22, 30, 36), and rabbit (23, 28) coronary rings. Also, feeding soy isoflavones to rhesus monkeys that have been fed an atherogenic diet resulted in vasodilation and increased blood flow through the coronary arteries (13, 27).

In the same controlled anesthetized animal preparation, the mechanisms of this coronary vasodilating effect were examined by repeating the experiment after blocking cholinergic and adrenergic receptors, the NO synthase and ER{alpha}/ERβ. We used atropine, phentolamine, and butoxamine to block cholinergic, {alpha}- and β2-adrenergic receptors at the same doses previously used in the same animal model to block the autonomic nervous system (6, 34, 41). Neither the blockade of cholinergic receptors nor the administration of phentolamine affected the coronary response to genistein, indicating that the above effect did not involve cholinergic or {alpha}-adrenergic receptors.

The results obtained after butoxamine administration showed the involvement of β2-adrenoceptors in the coronary effects elicited by intracoronary genistein; the β2-specific blocker completely prevented the genistein-induced increase of coronary blood flow. This finding is partly consistent with previous data revealing an interaction between genistein and β-adrenoceptors. For instance, in rat aortic rings, genistein potentiated the relaxation induced by isoproterenol, an effect that could be related to the inhibition of cAMP-phosphodiesterase activity (17). Furthermore, both cAMP and β-adrenoceptors have been related to the relaxing effect elicited by flavonoids on rat uterine smooth muscle (42).

The present findings also showed that the coronary vasodilation caused by intracoronary infusion of genistein involved both the endothelial release of NO, through β2-adrenoceptors, and its intracellular pathway. Although the actual release of NO was not measured in the pig, the regional vasodilation elicited by infusion of the genistein was abolished by the local intraarterial administration of L-NAME, which is known to inhibit the formation of NO (43). The dose of 2 mg for 1 ml/min of measured blood flow of the blocking agent used is similar to that previously shown in anesthetized pigs to abolish the coronary, mesenteric, renal, and iliac vasodilation caused by iv infusion of 17β-estradiol (6) and intraarterial infusion of testosterone (44) as well as the coronary vasoconstriction caused by iv infusion of prolactin and dehydroepiandrosterone (34, 45).

The results obtained regarding the role of NO in the vascular effect elicited by genistein are of particular interest. For instance, feeding soy isoflavones to rhesus monkeys that have been fed atherogenic diet resulted in vasodilation and increased blood flow in coronary arteries in situ through the operation of endothelial factors (13, 27). In contrast, other factors, more related to calcium movements or calcium-sensitive potassium channels than to endothelium, have been implicated in the genistein-induced vasodilation of isolated coronary arteries of their rings (23, 28, 29, 30, 46). On the other hand, genistein was found to reverse endothelial dysfunction of aortic rings after ovariectomy (18). In postmenopausal women, the therapy with this phytoestrogen increased plasma nitrites/nitrates levels and decreased plasma endothelin-1, thereby enhancing the ratio of NO oxidized products to endothelin-1 (19). Finally, genistein reduced the elevated blood pressure and endothelial dysfunction in spontaneously hypertensive rats (47); this latter effect appears to be related to increased eNOS activity.

The coronary effects elicited by genistein were abolished by the ER antagonist fulvestrant, also known as ICI 182–780, given in the coronary artery at doses corresponding to those used in rats to abolish the postconditioning effect induced by doses of the phytoestrogen similar to the ones used in the present paper (26). The same dose of fulvestrant was able in the same experimental model to abolish the coronary effects elicited by iv administration of 2 µg/h 17β-estradiol (data not shown). Because fulvestrant is not specifically selective of a particular ER subtype (48), our findings cannot be said to provide evidence against involvement of either the {alpha}- or β-subtype. However, because genistein has been reported to have up to 20- to 30-fold higher binding affinity, more for ERβ in the solid-phase binding assay study (49) and in the vasculature (50), it is considered possible that ERβ was involved rather than ER{alpha}. Thus, even whether the expression of ERβ in the coronary vasculature of pigs was not established, it can be assumed that ERβ plays a pivotal role in vascular protection and could be the main receptor that mediates the effects of phytoestrogen in the vascular wall as previously reported (50).

In vitro study
The results obtained in PAE cells confirm the ones obtained in the anesthetized pig. The administration of genistein at a concentration that in the present study induced the highest effects and similar to the one used in pigs and that was able to induce a late but sustained activation of the eNOS in human endothelial cells (51) acutely increased the NO production and level of phosphorylation of eNOS, ERK, p38, and Akt, which are involved in the intracellular signaling of NO production through β-adrenoceptors also in PAE cells (34, 37, 52). The effect on NO synthesis was prevented by the concomitant administration of L-NAME, wortmannin, UO126, and SB203580 using doses that are known to block the effect of prolactin in PAE cells (34) and similar to the ones previously used by others to block intracellular pathways related to ERK, p38, and Akt in endothelial cells (53, 54). Thus, the results obtained support the hypothesis of the activation of endothelial NO synthesis being linked to the vasodilation caused by genistein in the anesthetized pig and are in agreement with previous studies (13, 15, 16, 51). Furthermore, the findings obtained in the present study show the involvement of PI3K/Akt, ERK/MAPKs, and p38/MAPK pathways in the intracellular signaling leading to eNOS activation. These findings are in agreement with previous data obtained in human aortic and umbilical cells (55). Furthermore, the blockade of adenyl cyclase through 2'5' dideoxyadenosine at a concentration that was able to prevent the effect of the adenyl cyclase agonist forskolin and similar to the one used by others in endothelial cells (56) abolished the effects of genistein, thus confirming previous studies regarding the role of cAMP in the effects of the phytoestrogen (37).

In addition, the results obtained with β-agonists and antagonists confirmed the involvement the β2-adrenergic receptor-mediated effect related to the release of NO as mechanism of action of the vasodilation induced by genistein in the animal model. Thus, genistein potentiated the increased NO production caused by isoproterenol and salbutamol, an effect that was prevented by the pretreatment of PAE cells with L-NAME, wortmannin, UO126, and SB203580.

Interestingly the blockade of adenyl cyclase prevented both the effects of the isoproterenol and salbutamol, given alone and in costimulation with genistein, thus confirming the involvement of cAMP in the intracellular signaling induced by β2-adrenoceptor activation, leading to NO in endothelial cells (37).

These findings support the hypothesis of an interaction between genistein and β2-adrenoceptors, leading to eNOS activation and NO production through the involvement of PI3K/Akt, ERK/MAPKs, and p38/MAPK as intracellular pathways. This hypothesis can be further confirmed by the results obtained in experiments performed with butoxamine, which abolished the effect of genistein on NO production, and by the analysis of the level of phosphorylation of ERK1/2, Akt, and p38. It is noteworthy that these results are in agreement with in vitro studies evidencing an interrelation between flavonoids and β-adrenoceptors (31, 42).

The effect of genistein was prevented by the administration of ER blockers given at a dosage that prevented the response of PAE to 17β-estradiol and similar to the one previously shown to inhibit the effect on NO synthesis of equimolar doses of genistein (38). These findings are in agreement with the results obtained in the anesthetized pigs and with previous data regarding the role of ERs in the vascular effects elicited by genistein (26). Furthermore, the results obtained with isoproterenol or salbutamol, given alone or with genistein in the presence of fulvestrant, indicate a relationship between the β2-adrenoceptors and ERβ pathways, which is a new finding.

Taken together, this study identifies a novel mechanism of NO production through ER{alpha}/ERβ in which genistein initiates cytoplasmic signaling events upon binding to estrogenic receptor, leading to cAMP production and phosphorylation of factors such as ERK1/2, p38, and Akt, which ultimately induce activation of eNOS. It is quite interesting that among the above factors Akt is reported to act as integrator of different signal transduction pathways converging on eNOS, including endothelial β2-adrenergic receptors. Indeed the blockade of Akt in this study was found to prevent the effect of β2-agonist on NO production. Moreover, in the cascade of events, the induction of G proteins like G protein receptor 30, a seven-transmembrane receptor that has been implicated to mediate the nongenomic signaling of 17β-estradiol (57), or other factors involved in the β2-adrenergic signaling pathway (58), could represent the link among genistein, β-adrenergic system, and estrogenic receptors. Thus, the role of β2-adrenoceptors in the NO production caused by genistein could be rather related to the cytoplasmic events on estrogenic receptors than to a physical interaction with the phytoestrogen. By this way genistein could cause the β2-adrenergic activation of NO synthesis through ER{alpha}/ERβ.

Finally, it is important to point out that our findings regarding the genistein-induced coronary vasodilation are limited to an acute effect of this isoflavone phytoestrogen. However, it is known in animals that chronic supplementation of dietary genistein results in coronary vasodilation mediated by endothelial mechanisms (13, 27) and in relaxation of aortic rings that involved endothelial mechanisms in a manner similar to that of 17β-estradiol (18). In humans, chronic dietary intake of genistein was found to result in brachial artery vasodilation that involved endothelium-dependent mechanisms attributed to an increased ratio of nitric oxide to endothelin (19). Therefore, these considerations indicate that our findings could have provided a mechanism for the observed beneficial cardiovascular effects of phytoestrogens (10, 11, 12).

In conclusion, our results have shown that the local intracoronary administration of genistein in the pig primarily increased coronary blood flow through a β2-adrenergic mediated increase in NO release. The coronary effects of genistein are related to ER{alpha}/ERβ and involve the ERK1/2, Akt, and p38 pathways leading to eNOS phosphorylation.


   Acknowledgments
 
We thank the Azienda Ospedaliera Maggiore della Carità di Novara for its help.


   Footnotes
 
This work was supported by grants from the Università del Piemonte Orientale.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 17, 2008

Abbreviations: DAF-FM, Diaminofluorescein; DMSO, dymethilsulfoxide; dP/dtmax, maximum rate of change of left ventricular systolic pressure; eNOS, endothelial NO synthase; ER, estrogen receptor; L-NAME, N{omega}-nitro-L-arginine methyl ester; NO, nitric oxide; PAE, porcine aortic endothelial; PI3-K, phosphatidylinositol 3-kinase.

Received October 3, 2007.

Accepted for publication January 9, 2008.


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 Materials and Methods
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 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals