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CARDIOVASCULAR

Effects of Phytoestrogens on Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a and Ca²⁺ Uptake into Cardiac Sarcoplasmic Reticulum

Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada (M.L.O., M.E.K., G.J.K.); Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts (T.W.H.); and Department of Physiology, Queens University, Kingston, Ontario, Canada (C.A.W.)

Received July 19, 2005; accepted October 13, 2005.

	Abstract

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Phytoestrogens are naturally occurring estrogenic compounds found in plants and plant products. These compounds are also known to exert cellular effects independent of their interactions with estrogen receptors. We studied the effects of the phytoestrogens phloretin, phloridzin, genistein, and biochanin A on Ca²⁺ uptake into the cardiac muscle sarcoplasmic reticulum (SR). Genistein and biochanin A did not affect SR Ca²⁺ uptake. On the other hand, phloretin and phloridzin decreased the maximum velocity of SR Ca²⁺ uptake but did not affect the Hill coefficient or the Ca²⁺ sensitivity of uptake. Measurements of the ATPase activity of the cardiac SR Ca²⁺ pump (SERCA2a) revealed direct inhibitory effects of phloretin and phloridzin on SERCA2a. Neither compound induced a detectable change in the permeability of the SR membrane to Ca²⁺. These results indicate that phloretin and phloridzin inhibit cardiac SR Ca²⁺ uptake by directly inhibiting SERCA2a.

In previous studies (Kargacin et al., 2000; Dodds et al., 2001), we showed that the mixed estrogen antagonists/agonists tamoxifen, 4-hydroxytamoxifen, and clomiphene inhibit Ca²⁺ uptake into cardiac sarcoplasmic reticulum (SR). Inhibition of Ca²⁺ uptake by tamoxifen was not mimicked by -estradiol and -estradiol did not alter the effects of tamoxifen (Kargacin et al., 2000; Dodds et al., 2001), indicating that the action of tamoxifen is not mediated by interaction with estrogen receptors. In previous work with phytoestrogens, it was shown (Shoshan and MacLennan, 1981) that quercetin directly inhibits the ATPase activity of the SR Ca²⁺ pump. These results raise the possibility that estrogenic compounds in general may directly affect SR function. This possibility was also suggested by Liew et al. (2003) who showed that the phytoestrogen genistein increases cell shortening and the amplitude of electrically evoked Ca²⁺ transients in isolated guinea pig cardiac myocytes in spite of the fact that it inhibited L-type Ca²⁺ currents. From these results, and comparison of caffeine-evoked Ca²⁺ transients in Na⁺-free solution in the presence and absence of genistein, the authors of the study concluded that the SR Ca²⁺ load was increased in the cells; however, direct effects of genistein on SR Ca²⁺ uptake were not examined. We were thus motivated to examine, directly, the effects of genistein, its parent compound biochanin A, and other phytoestrogens on Ca²⁺ uptake into cardiac SR vesicles. Here, we report results of experiments with biochanin A, genistein, phloretin, and phloridzin.

	Materials and Methods

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Materials. Sucrose was purchased from MP Biomedicals (Aurora, OH). Oxalic acid and MgCl₂ were purchased from Fluka (Ronkonkoma, NY). KCl, K₂ATP, Na₂ATP, CaCl₂, HEPES (K⁺ salt), creatine phosphate, creatine phosphokinase, NADH, pyruvate kinase, phosphoenolpyruvate, histidine, genistein, biochanin A, phloretin, phloridzin (phlorizin dihydrate), and apyrase (grade VII) were purchased from Sigma-Aldrich (St. Louis, MO). Lactate dehydrogenase was purchased from Worthington Biochemicals (Freehold, NJ). Fura-2 (free acid), Ca²⁺ calibration kits (calcium calibration buffer kit 2), and 4-bromo A-23187 (4-bromocalcimycin) were purchased from Invitrogen (Carlsbad, CA). Unless otherwise noted, experiments were carried out at 22°C. Results are expressed as ±1 S.E.

Preparation of Membrane Vesicles. SR vesicles were prepared from canine cardiac ventricular tissue as described by Chamberlain et al. (1983) except that the sucrose gradient centrifugation step was omitted (Kargacin and Kargacin, 1994). Vesicles were stored at -80°C in a buffer containing 300 mM sucrose, 100 mM KCl, and 10 mM histidine, pH 7. Animals were euthanized in accordance with protocols accepted by the Canadian Council on Animal Care and the National Institutes of Health.

Measurement of SR Ca²⁺ Uptake. Ca²⁺ uptake into cardiac SR vesicles was measured with fura-2 as described previously (Kargacin and Kargacin, 1994, 1998a,b, 2000; Dodds et al., 2001) using either a SPEX CMX fluorimeter (SPEX Industries, Edison, NJ) or a fluorimeter (Photon Technologies International, Lawrenceville, NJ) with the excitation wavelength alternated between 340 and 380 nm. Fluorescence emission was measured at 510 nm; 340/380 fluorescence ratios were determined every second. SR Ca²⁺ uptake was measured in uptake buffer containing 100 mM KCl, 4 mM MgCl₂, 20 mM K-HEPES, 1.35 mM ATP (K⁺ or Na⁺ salt), and 2.9 µM fura-2 free acid, pH 7.0 (Kargacin et al., 1998a). To maintain a supply of ATP for the SR Ca²⁺ pump, an ATP-regenerating system consisting of 1.35 mM creatine phosphate and 1.6 U/ml creatine phosphokinase was added to the uptake buffer. Measurements were made in a stirred cuvette containing 2 ml of buffer. For most experiments, 10 mM oxalate was included in the uptake buffer. Oxalate crosses the SR membrane and precipitates Ca²⁺ in the SR lumen, thereby reducing the luminal [Ca²⁺]_free. This permits greater unidirectional Ca²⁺ movement and prolongs the initial rapid phase of uptake (Martonosi and Feretos, 1964). Some experiments were also conducted without oxalate in the uptake buffer. Stock solutions of each estrogenic compound were prepared in 95% ethanol/5% H₂O so that the same fluid volume (2 µl) was added to the cuvette for each experiment to achieve the desired final concentration of the compound being tested. In control experiments (without the compounds), 2 µl of 95% ethanol (0.1% final concentration) was added to the uptake buffer. This did not affect the kinetics of SR Ca²⁺ uptake. Uptake kinetics were also not affected when Na₂ATP replaced K₂ATP in the uptake buffer.

Before each experiment, light scatter (excitation 340 and 380 nm, emission 510 nm) was measured for 30 s in uptake buffer containing SR vesicles and all chemical components except fura-2, ATP, and the ATP-regenerating system. Fura-2, ATP, and the ATP-regenerating system were then added to the cuvette. Ca²⁺ was added from a 2.5 mM stock solution to bring the starting [Ca²⁺]_free in the cuvette to 3 to 4 µM. As Ca²⁺ is taken up by the vesicles, extravesicular [Ca²⁺] declines and results in a decrease in the 340/380 fluorescence ratio of fura-2.

Fura-2 Calibration and Determination of [Ca²⁺]_free, [Ca²⁺]_total, and Uptake Rate. Curves of Fura-2 340 and 380 fluorescence intensity, plotted as a function of time, were corrected for light scatter and then used to calculate 340/380 ratio (R) versus time curves. The [Ca²⁺]_free was determined at each time point from the 340/380 fluorescence measurements according to the following equation (Grynkiewicz et al., 1985),

(1)

where R_max is the fura-2 340/380 fluorescence ratio measured in saturating Ca²⁺ buffer (2.5 mM CaCl₂) and the R_min is the 340/380 ratio measured in Ca²⁺-free solution (25 mM EGTA in uptake buffer). K_d, the dissociation constant for Ca²⁺, is 200 nM (Williams et al., 1987). is measured with 380-nm excitation and 510-nm emission and is the ratio of fura-2 fluorescence intensity measured in Ca²⁺-free buffer to that measured in saturating Ca²⁺ buffer. The total calcium concentration ([Ca²⁺]_total) in the extravesicular solution in the cuvette was determined at each time point from the [Ca²⁺]_free as described previously by taking into account the Ca²⁺ binding to various buffer components (Kargacin and Kargacin, 1994).

Calcium uptake velocity (micromoles per minute per milligram) was calculated at each time point from the negative derivative of the [Ca²⁺]_total versus time curves using the following equation,

(2)

where A (= v/w) is the volume of solution in the cuvette divided by the amount of SR protein in the cuvette. The velocity values at each time point were then plotted against the corresponding [Ca²⁺]_free values. Maximum uptake velocity (V_max), [Ca²⁺] at half-maximal velocity ([Ca²⁺]_50%), and the Hill coefficient (n_H) of Ca²⁺ uptake were determined from these plots by fitting them to the Hill equation as described previously (Kargacin and Kargacin, 1994). V_max, expressed as micromoles of Ca²⁺ per minute per milligram of SR protein, is dependent on the amount of Ca²⁺-ATPase relative to the total amount of protein present in a vesicle preparation and can, therefore, vary for different vesicle preparations. To compensate for this variation, V_max values are given as a percentage of the average V_max value for control experiments from the same vesicle preparation. This allows results from different vesicle preparations to be compared. In the control experiments reported here, V_max values obtained from different vesicle preparations ranged from 0.180 to 0.460 µmol Ca²⁺ · min^-1 · mg^-1. Uptake was completely inhibited by 5 to 10 µM thapsigargin.

Calibration of Fura-2 Fluorescence Intensity in the Presence of Phytoestrogens. Many phytoestrogens absorb light over the same wavelength range as fura-2 (Escarpa and Gonzalez, 1998). Figure 1A shows absorbance spectra for phloretin in uptake buffer at concentrations ranging from 5 to 50 µM. Similar absorbance spectra were obtained for the other phytoestrogens (genistein, biochanin A, and phloridzin) used in this study. For each compound, the absorbance was greater at shorter wavelengths reducing the excitation intensity for fura-2 at 340 nm more than at 380 nm (Fig. 1B), thus affecting the calibration parameters R_min and R_max. R_min and R_max were, therefore, measured for each concentration of phytoestrogen tested. The calibration parameter was not different in the absence or presence of the compounds tested. Ca²⁺ calibration kits (see Materials and Materials), containing a series of buffers with different [Ca²⁺]_free values, were used to show that these compounds did not affect the K_d for Ca²⁺ binding to fura-2 (see eq. 1). These results show that the estrogenic compounds we used simply absorb light at wavelengths 450 nm but do not alter fura-2 fluorescence emission or its Ca²⁺ binding properties.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Phloretin absorbance and its effect on fura-2 and NADH fluorescence intensity. A, absorbance as a function of wavelength in the presence of 5 (black), 10 (red), 20 (green), 30 (yellow), 40 (blue), and 50 µM phloretin (magenta). B, fura-2 fluorescence intensity at 510 nm was measured as a function of excitation wavelength for Ca2+ calibration buffers in the absence (black line, Rmax buffer; dashed black line, Rmin buffer) and presence of 50 µM phloretin (red line, Rmax buffer + phloretin; dashed red line, Rmin buffer + phloretin). C, NADH fluorescence (350-nm excitation, 450-nm emission) as a function of NADH concentration in the absence (black line) and in the presence of 50 µM phloretin (red line).

(3)

(4)

(5)

where PEP is phosphoenolpyruvate, PK is pyruvate kinase, and LDH is lactate dehydrogenase. The rate of decline in NADH fluorescence (excitation 350 nm; emission 450 nm) is proportional to the rate of ATP hydrolysis by the SR Ca²⁺ pump. To measure Ca²⁺-ATPase activity, ATP (final concentration 1 mM) was added to a cuvette containing 2 ml of ATPase buffer (100 mM KCl, 4 mM MgCl₂, 1.5 µM free-Ca²⁺, 1 mM EGTA, 0.42 U/ml PK, 0.9 mM PEP, 4.4 U/ml LDH, and 20 mM HEPES, pH 7.0) with 90 µM NADH and either 0.1% ethanol (final concentration) or 100 µM of one of the phytoestrogens. For the measurements of ATPase activity, the Ca²⁺ ionophore 4-bromo A-23187 (3.3 µM) was added to prevent a Ca²⁺ gradient from forming across the SR membrane and from affecting the turnover of the pump. This allows direct effects on SERCA to be determined. After allowing any contaminating ADP to react with the assay components (eqs. 4 and 5), additional NADH was added, if necessary, to bring the NADH concentration up to 90 µM. At this point, cardiac SR vesicles were added, and the rate of decline in NADH fluorescence was determined.

Background light scatter (measured in the presence of all components except NADH) and oxidation of NADH by sample components (measured in the presence of all components except ATP) were determined and used to correct the measurements of the ATPase activity of the SR Ca²⁺ pump (Kargacin et al., 2000).

Calibration curves relating NADH fluorescence intensity to NADH concentration were obtained by adding known concentrations of NADH to uptake buffer. The calibration curves were fit using the following exponential equation (Kargacin et al., 2000).

(6)

All of the estrogenic compounds tested in this study absorbed light at 350 nm, as is shown for 50 µM phloretin in Fig. 1A. Therefore, calibration curves relating NADH fluorescence to NADH concentration were determined for each phytoestrogen concentration used in the experiments (Fig. 1C). In the preparations that were used for this study, 78 ± 3% (n = 15) of the ATPase activity could be inhibited by thapsigargin (10 µM) and was thus attributable to the SR Ca²⁺ pump. This result is similar to those obtained in our previous study (Kargacin et al., 2000). All measurements of ATPase activity were adjusted for the thapsigargin-insensitive activity (see Results).

Experiments were also carried out to demonstrate that the estrogenic compounds used in this study did not affect the reactions in the enzyme-coupled assay described by eqs. 4 and 5. To do this, 100 µM of the estrogenic compound being studied (or vehicle) was added to 2 ml of ATPase buffer. Measurements were made using 350-nm excitation and 450-nm emission. After measurement of background light scatter, NADH (final concentration 90 µM) was added to the cuvette followed by ADP (final concentration 0.34 mM). Conversion of ADP to ATP and NADH to NAD⁺, in the reactions described by eqs. 4 and 5, resulted in a decline in NADH fluorescence with time. Plots of NADH fluorescence versus time were converted to plots of [NADH] as a function of time using calibration curves as described above. As shown in Table 1, the rate of decline in [NADH] determined when ADP was added to the buffer was not altered by the presence of phloretin or phloridzin. Thus, these compounds do not affect reactions 4 and 5.

Measurement of Passive Ca²⁺ Release. To examine changes in the permeability of the SR membrane to Ca²⁺, SR vesicles were loaded as described above with Ca²⁺ in uptake buffer without oxalate. To initiate uptake in these experiments, ATP was added without the ATP-regenerating system. Uptake was monitored with fura-2 to ensure that vesicle samples to be compared were loaded to the same extent. After uptake was complete and the system had reached equilibrium, ethanol (0.1%, vehicle only control), phloretin (100 µM final concentration), or phloridzin (100 µM final concentration) was added to the vesicles. After an additional 60 s, apyrase (10 units/ml), was added to deprive the SR Ca²⁺ pump of its energy supply. The ATPase and ADPase activities of apyrase deprive the pump of ATP and ADP, thereby preventing it from pumping Ca²⁺ into the SR and from running in reverse mode and actively extruding Ca²⁺ from the SR. Thus, in the presence of apyrase, the SR passively releases loaded Ca²⁺ and the rate of this release is a measure of the permeability of the SR membrane to Ca²⁺. Control experiments were conducted to determine the concentration of apyrase required for the release experiments. Concentrations of apyrase >10 U/ml did not increase the rate or extent of Ca²⁺ release from Ca²⁺-loaded SR vesicles over that measured at 10 U/ml apyrase, indicating that the enzyme was not rate limiting at a concentration of 10 U/ml. The ability of apyrase to completely remove ADP from the uptake buffer during the release experiments was also examined. After SR vesicles were Ca²⁺ loaded and Ca²⁺ release was induced with apyrase, NADH, PEP, and PK were immediately added, and changes in NADH fluorescence were measured. The presence of ADP in the uptake buffer would result in a decrease in NADH fluorescence (see eqs. 4 and 5). In these control experiments, ADP was not detectable in the uptake buffer after application of apyrase.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Measurement of cardiac SR Ca2+ uptake in the presence of genistein. A, extravesicular [Ca2+]free as a function of time for an experiment done in the presence (closed circles) and in the absence (open circles) of 20 µM genistein. Note that because extravesicular [Ca2+] is measured in these experiments, uptake of Ca2+ into the SR results in a decrease in [Ca2+]free with time. B, extravesicular [Ca2+]total as a function of time for the experiment in A. The Vmax for uptake was determined from velocity versus [Ca2+]free curves as described under Materials and Methods; for the data shown in A and B control Vmax = 0.37 µmol · min-1 · mg-1 and Vmax in the presence of 20 µM genistein = 0.36 µmol · min-1 · mg-1. C, summary of results with genistein. Results in C are expressed as percentage of the mean Vmax for control experiments conducted in the absence of genistein. Vmax values in C were not significantly different from control (p > 0.05). Note that in A and B, only every fifth data point is plotted, and the curves were offset slightly along the time axis for clarity; 25 µg of SR vesicle protein was used in all experiments.

	Results

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Figure 3A shows extravesicular [Ca²⁺]_total plotted as a function of time for a control experiment and an experiment done in the presence of 20 µM phloretin. In this experiment, phloretin reduced the V_max of SR Ca²⁺ uptake into cardiac SR vesicles to 71% of control. Figure 3B shows the maximal velocity of calcium uptake (V_max) as a percentage of control for different concentrations of phloretin. Phloretin reduced the V_max of SR Ca²⁺ uptake between the concentrations of 10 and 200 µM. Half-maximal inhibition was at 60 µM. At the highest concentration of phloretin used (200 µM), V_max was 21.1 ± 0.4% (n = 3) of control. In contrast to these results, phloridzin did not significantly reduce the V_max of SR Ca²⁺ uptake at concentrations lower than 50 µM; however, V_max was decreased at higher concentrations (Fig. 3C). At the highest concentration of phloridzin used (200 µM), V_max was reduced to 75 ± 4% (n = 6) of control. There were no significant changes in the Hill coefficient or the Ca²⁺ sensitivity of SR Ca²⁺ uptake in the presence of either phloretin or phloridzin (data not shown). We also examined the effects of phloretin and phloridzin at 37°C. At the higher temperature, the effect of phloretin on V_max was less than that seen at room temperature and the effect of phloridzin was greater. At 37°C, V_max was reduced to 65 ± 1% (n = 4) of control in the presence of 100 µM phloretin and to 53 ± 1% (n = 4) of control in the presence of 100 µM phloridzin.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Inhibition of cardiac SR Ca2+ uptake by phloretin and phloridzin. A, extravesicular [Ca2+]total as a function of time for an experiment done in the presence (closed circles) and in the absence (open circles) of 20 µM phloretin. The Vmax for uptake was determined from velocity versus [Ca2+]free curves as described under Materials and Methods; for the data shown in A, control Vmax = 0.38 µmol · min-1 · mg1- and Vmax in the presence of 20 µM phloretin = 0.27 µmol · min-1 · mg-1. Note that in A, only every fifth data point is plotted for clarity. B and C, effects of phloretin and phloridzin on the Vmax of cardiac SR Ca2+ uptake. B, inhibition of Vmax of cardiac Ca2+ uptake by phloretin. C, inhibition of Vmax of Ca2+ uptake in the presence of phloridzin. Results in B and C are expressed as percentage of the mean Vmax for control experiments conducted in the absence of the drugs. Vmax values in B are significantly different from control (at p 0.05) for [phloretin] 20 µM; Vmax values in C are significantly different from control for [phloridzin] 50 µM. In B, n = 7 for all [phloretin] except 0 µM (n = 38), 40 µM (n = 5), 100 µM (n = 8), 150 µM (n = 4), and 200 µM (n = 3). In C, n = 3 for all [phloridzin] except 0 µM (n = 21), 100 µM (n = 8), and 150 µM (n = 4). Uptake was measured with 25 µg of SR vesicle protein in the cuvette in all experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Effects of phloretin on SR Ca2+ uptake when the [Ca2+]free gradient across the SR membrane was allowed to increase. Cardiac SR vesicles (25 µg of vesicle protein) were loaded during three Ca2+ additions. Before the third Ca2+ addition, 2 µl of 95% ethanol (red trace; vehicle only control) or 100 µM phloretin (blue trace) was added. Maximum uptake velocity following the third Ca2+ addition was 0.21 µmol · min-1 · mg-1 for the control experiment and 0.08 µmol · min-1 · mg-1 in the presence of phloretin.

Measurement of the ATPase Activity of the Cardiac SR Ca²⁺ Pump in the Presence of Phloretin and Phloridzin. The rate at which Ca²⁺ is taken up into cardiac SR vesicles could be affected by phloretin or phloridzin if these compounds directly inhibit the SR Ca²⁺ pump. To test this possibility, the ATPase activity of SERCA2a in cardiac SR vesicles was measured using the enzyme coupled ATPase assay described by eqs. 3 to 5 under Materials and Methods. For these experiments, ATPase activity was measured before and after phloretin (100 µM, final concentration), phloridzin (100 µM, final concentration), or ethanol only (0.1%, final concentration; vehicle control) was added to the vesicles. Results of one experiment with phloretin are shown in Fig. 5A; Fig. 5B summarizes the results of the phloretin experiments. The ATPase activity of the SR Ca²⁺ pump was reduced to 67 ± 3% (n = 12) of control in the presence of 100 µM phloretin. Addition of 0.1% ethanol only did not affect the ATPase activity of the vesicle preparations. As noted under Materials and Methods, approximately 20% of the ATPase activity of our SR vesicle preparations was thapsigargin insensitive (Fig. 5B). The thapsigargin-insensitive activity was unaltered by phloretin or phloridzin, indicating that only the Ca²⁺ pump activity of our preparations was affected. When the results of the ATPase measurements in the presence of phloretin were corrected for the thapsigargin-insensitive activity, the ATPase activity attributable to the cardiac SR Ca²⁺ pump was reduced to 57 ± 9% of control by 100 µM phloretin. Phloridzin (100 µM) also significantly decreased the ATPase activity of cardiac SR Ca²⁺ pump but to a lesser extent than phloretin (Fig. 5B). ATPase activity in the presence of 100 µM phloridzin was 84 ± 2% (significantly different at p < 0.0034; n = 7) of that measured in control experiments (ethanol only added). When this value was corrected for the thapsigargin-insensitive ATPase activity, the thapsigargin-sensitive ATPase activity was 78 ± 8% of control in the presence of 100 µM phloridzin.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Measurement of the ATPase activity of the cardiac SR Ca2+ pump in the presence of phloretin or phloridzin. A, SERCA2a ATPase activity (measured as a change in [NADH]; see Materials and Methods) before, and following, the addition of phloretin (100 µM) to cardiac SR vesicles. B, summary of results of ATPase assay experiments. Phloretin, phloridzin, and thapsigargin significantly decreased ATPase rates (p < 10-6, n = 12 for phloretin; p < 0.004 for phloridzin, n = 7; and p < 10-6, n = 15 for thapsigargin) over control values. Results are plotted as percentage of the mean rate of ATP turnover following the addition of ethanol only (0.1% final concentration) to the assay; the mean ATPase rate following the addition of ethanol to the vesicles was not significantly different from that observed before its addition (p = 0.17; n = 16). SR protein (60 µg) was used for the experiments shown.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Phloretin and phloridzin do not increase the passive Ca2+ permeability of SR vesicles. In A and B, extravesicular [Ca2+]total is shown as a function of time. SR vesicles (80 µg of vesicle protein) were first allowed to take up Ca2+ until equilibrium was reached before 0.1% ethanol (A) or 100 µM phloretin (B) was added to the uptake buffer. After an additional 60 s, 10 units/ml apyrase was added to deprive the SR Ca2+ pump of ATP and ADP (see text for details). The initial Ca2+ release rate was determined from a linear fit to the first 10 s of release.

	Discussion

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The main findings of this study are as follows. We have found that the phytoestrogens genistein and biochanin A at concentrations 30 µM are without effect on cardiac SR Ca²⁺ uptake. We have shown that phloretin (10-200 µM) and phloridzin (50-200 µM) reduce the V_max of Ca²⁺ uptake into cardiac SR. Phloretin and phloridzin were also shown to directly inhibit the ATPase activity of SERCA2a. Neither compound affects the rate at which Ca²⁺ is passively released from Ca²⁺-loaded SR vesicles, indicating that they do not alter the Ca²⁺ permeability of the SR membrane.

Inhibition of SERCA2a by phloretin and phloridzin is consistent with reports of their effects on other ATP-dependent enzymes. The mitochondrial F0F1-ATPase/ATP synthase of rat brain is inhibited by phloretin, phloridzin, genistein, and biochanin A and a number of other phytoestrogens (Zheng and Ramirez, 2000). The ATPase activity of the F0F1 synthase was inhibited by 40% at 70 µM phloretin and by 14% in the presence of 70 µM phloridzin (Zheng and Ramirez, 2000). The rat liver plasma membrane Ca²⁺ pump is inhibited by several phytoestrogens, including phloretin (to about 50% of control in 100 µM phloretin; Thiyagarajah et al., 1991). Phloridzin, however, did not affect the latter Ca²⁺ pump (Thiyagarajah et al., 1991).

The mechanism by which phloretin and phloridzin inhibit the ATPase activity of SERCA2a (and some other ATPases) is not known, although our experiments and those of others can rule out some possibilities. The fact that neither [Ca²⁺]_50% nor n_H for SR Ca²⁺ uptake was affected by either compound indicates that Ca²⁺ binding to SERCA2a was not altered. The ability of phloretin and phloridzin, but not genistein or biochanin A, to inhibit SR Ca²⁺ uptake does not correlate with their estrogenic potency. The order of potency of the compounds for the -estrogen receptor subtype is genistein > phloretin > biochanin A, and for the -receptor subtype is genistein > biochanin A > phloretin (Kuiper et al., 1998). At the highest concentration tested (30 µM), genistein did not affect SR Ca²⁺ uptake, whereas phloretin, at the same concentration, inhibited uptake to approximately 70% of control. It was also shown in a previous study (Dodds et al., 2001) that -estradiol does not affect Ca²⁺ uptake into canine cardiac SR vesicles nor does it alter the inhibitory effect of the antiestrogen tamoxifen on Ca²⁺ uptake. It is thus unlikely that the effects of phloretin (or phloridzin) on SR Ca²⁺ uptake are mediated through estrogen receptors.

It has been hypothesized that phloretin and phloridzin can affect membrane proteins and the movement of hydrophobic ions across lipid monolayers and bilayers by altering the intrinsic dipole potential of membranes (Reyes et al., 1983; Franklin and Cafiso, 1993; Sukhorukov et al., 2001; Valenta et al., 2004). Phloretin is dipolar and is thought to insert into membranes with its dipole orientated in a direction that reduces the intrinsic membrane dipole potential (Reyes et al., 1983; Franklin and Cafiso, 1993; Sukhorukov et al., 2001; Valenta et al., 2004). Phloridzin has also been shown to interact with some model membranes in a manner similar to that of phloretin (Valenta et al., 2004). The effects of phloretin on the membrane dipole are opposite to those of 6-ketocholestanol, which is thought to increase the dipole potential of bilayers (Reyes et al., 1983; Franklin and Cafiso, 1993; Sukhorukov et al., 2001). If the effects of phloretin and phloridzin on SERCA2a are brought about by a decrease in the membrane dipole potential, one might expect 6-ketocholestanol to have the opposite effect on SERCA2a ATPase activity. We found, however, that 6-ketocholestanol (25-50 µM) does not affect the ATPase activity of SERCA2a (M. L. Olson, G. J. Kargacin, and M. E. Kargacin, unpublished data). Therefore, we cannot confirm or rule out the possibility that SERCA2a activity is affected by the membrane dipole potential.

It is interesting that increasing the temperature to 37°C affected the extent to which phloretin and phloridzin inhibited Ca²⁺ uptake velocity differently (the effect of phloretin on V_max was less at 37°C than it was at room temperature, whereas the effect of phloridzin on V_max was greater at 37°C than it was at room temperature). As noted, both compounds are thought to affect the membrane dipole potential; however, the extent to which they insert into membranes may be differentially affected by temperature and/or temperature-dependent changes in membrane fluidity. It is also possible that the mechanism by which the compounds inhibit SERCA activity (e.g., direct binding versus indirect effects because of their insertion into, and interaction with, the lipid bilayer) is affected differently by temperature.

The extent to which the maximum velocity of SR Ca²⁺ uptake was inhibited by phloretin (60% inhibition) in our experiments is greater than the extent to which phloretin inhibited the ATPase activity of SERCA2a (50% inhibition). Thus, although the effect of phloretin on SR Ca²⁺ uptake seems to be primarily because of direct inhibition of SERCA2a, it is possible that other factors are also involved in the inhibition of uptake. One mechanism by which phloretin could reduce the V_max of SR Ca²⁺ uptake in addition to inhibiting SERCA2a activity could be through the inhibition of the compensatory Cl^- and/or K⁺ movement that is thought to accompany Ca²⁺ uptake to maintain the electroneutrality of the SR membrane (for review, see Tada and Kadoma, 1995). Phloretin and phloridzin have structural similarity to the stilbene-derivative Cl^- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (Fan et al., 2001), and it has been shown that phloretin inhibits volume-sensitive (IC₅₀ of 30 µM) and cAMP-activated Cl^- channels (at concentrations greater than 100 µM) in human epithelial and mouse mammary cells (Fan et al., 2001). Phloridzin (100 µM), however, had no effect on these currents (Fan et al., 2001). Phloretin has also been reported to inhibit some types of K⁺ channels (Spires and Begenisich, 1989; Klusemann and Meves, 1991, 1992; Koh et al., 1994). Further technical developments facilitating the measurement of membrane potential in intracellular organelles will be required to better understand the actions of phloretin on cardiac SR Ca²⁺ uptake.

In a recent study (Liew et al., 2003), it was reported that genistein increased cell shortening and the Ca²⁺ load in guinea pig ventricular myocytes. The latter action could be partially explained by an inhibitory effect on the cardiac Na⁺/Ca²⁺ exchanger, and as a consequence, increased Ca²⁺ uptake into the SR. The authors of the study also speculated that genistein could increase SERCA-mediated SR Ca²⁺ uptake but did not test this possibility. Our results showing that neither genistein nor biochanin A affected SR Ca²⁺ uptake argue against the latter possibility. Our results with phloretin and phloridzin suggest that they also could affect the contractility of intact cardiac myocytes. In preliminary experiments (M. L. Olson, M. E. Kargacin, G. J. Kargacin, and C. A. Ward, unpublished data), we examined the effects of phloretin and phloridzin on electrically evoked Ca²⁺ transients in intact rat cardiac myocytes. Phloretin (100 µM) caused the myocytes to hypercontract, preventing us from further analyzing the Ca²⁺ transients in the cells. The area of the electrically evoked transients was significantly decreased in the presence of 100 µM phloridzin (transient area was 80 ± 7% of control; n = 6; p = 0.012). The latter result is consistent with an inhibition of SR Ca²⁺ uptake and a decrease in the SR Ca²⁺ load. The effects of phloretin are indicative of a serious alteration of Ca²⁺ homeostasis but further experimentation is necessary to determine the mechanism(s) involved.

It is interesting that a number of estrogenic compounds, including phloretin and genistein, seem to be cardioprotective. We and others (Liew et al., 2003) have demonstrated effects of these compounds on Ca²⁺-handling proteins involved in Ca²⁺ regulation in cardiac myocytes; however, a number of specific effects have been identified, and additional experimental work and a better understanding of the genomic and nongenomic actions of these compounds will be necessary to determine whether there is a unifying mechanism to explain their cardioprotective effects.

	Acknowledgements

 
We thank Teresa Emmett, Dragana Ponjevic, and Erwin Wirch for technical assistance.

	Footnotes

 
This work was supported by the Canadian Institutes of Health Research, the American Heart Association, the Heart and Stroke Foundation of Alberta, the Heart and Stroke Foundation of Ontario, and the Carol M. Yates Memorial Endowment for Cancer Research. C.A.W. is a Heart and Stroke Foundation of Canada Research Scholar, and M.L.O. is a Heart and Stroke Foundation of Canada Graduate Scholar.

doi:10.1124/jpet.105.092940.

ABBREVIATIONS: SR, sarcoplasmic reticulum; 4-bromo A-23187, 4-bromocalcimycin; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; [Ca²⁺]_free concentration of free Ca²⁺; [Ca²⁺]_total, total Ca²⁺, concentration; [Ca²⁺]_50%, Ca²⁺ concentration at half-maximal velocity; PEP, phosphoenolpyruvate; PK, pyruvate kinase; LDH, lactate dehydrogenase.

Address correspondence to: Dr. Gary Kargacin, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Dr. N.W., Calgary, AB T2N 4N1, Canada. E-mail: kargacin{at}ucalgary.ca

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