Effects of anti-oestrogens and beta-estradiol on calcium uptake by cardiac sarcoplasmic reticulum

Abstract

1. Tamoxifen and a group of structurally similar non-steroidal, triphenolic compounds inhibit the oestrogen receptor. In addition to this action, these anti-oestrogens are known to inhibit some types of plasma membrane ion channels and other proteins through mechanisms that do not appear to involve their interactions with the estrogen receptor but could be the result of their effect on membrane lipid structure or fluidity. 2. We studied the effects of beta-estradiol and three anti-oestrogens (tamoxifen, 4-hydroxytamoxifen and clomiphene) on Ca(2+) uptake into sarcoplasmic reticulum (SR) vesicles isolated from canine cardiac ventricular tissue. 3. The antiestrogens all inhibit SR Ca(2+) uptake in a concentration-dependent manner (order of potency: tamoxifen > 4-hydroxytamoxifen > or = clomiphene). Although these compounds rapidly inhibit net Ca(2+) uptake they do not have a similar rapid effect on the ATPase activity of the SR Ca pump. beta-estradiol has no effect on Ca(2+) uptake nor does it alter the inhibitory action of tamoxifen on the SR. 4. The differences in the effects of beta-estradiol and the anti-oestrogens on cardiac SR Ca(2+) uptake do not correlate with differences in the ways in which these compounds have been reported to interact with membrane lipids. Our results are consistent, however, with direct effects on a membrane protein (possibly an SR Cl(-) or K(+) channel).

Figure 1

Inhibition of cardiac SR Ca²⁺ uptake by tamoxifen and 4-hydroxytamoxifen. (A) 340/380 fluorescence ratio as a function of time after correction for light scattering and background fluorescence in a control experiment (open symbols) and in the presence of 5 μM tamoxifen (closed symbols). Note: because extravesicular [Ca²⁺] is measured in these experiments, uptake of Ca²⁺ into the SR is shown by a decrease in 340/380 fluorescence. (B) Extravesicular [Ca²⁺]_free as a function of time for the experiment in (A). (C) Extravesicular [Ca²⁺]_free as a function of time for a control experiment (open symbols) and an experiment done in the presence of 5 μM 4-hydroxytamoxifen (closed symbols). The V_max, for uptake was determined from velocity versus [Ca²⁺]_free curves as described in Methods and were: for the experiment in (A) and (B), control V_max=0.534 μmol min⁻¹ mg⁻¹, V_max in tamoxifen=0.122 μmol min⁻¹ mg⁻¹; for the experiment in (C), control V_max=0.586 μmol min⁻¹ mg⁻¹, V_max in 4-hydroxytamoxifen=0.312 μmol min⁻¹ mg⁻¹. Note: in (A – C) only every 5th data point is plotted for clarity; 25 μg of SR vesicle protein was used for each experiment.

Figure 2

Effects of tamoxifen, 4-hydroxytamoxifen and clomiphene on the V_max of cardiac SR Ca²⁺ uptake. (A) Inhibition of V_max of cardiac SR Ca²⁺ uptake by tamoxifen. (B) Inhibition of V_max by 4-hydroxytamoxifen. (C) Inhibition of V_max by clomiphene. Results in (A – C) are expressed as per cent of the mean uptake velocity for control experiments (shown by the open circles at the left in each plot) conducted in the absence of the drugs. Uptake was measured with 25 μg of SR vesicle protein in the cuvette; the number above each point indicates the number of experiments.

Figure 3

Addition of tamoxifen, 4-hydroxytamoxifen or clomiphene to cardiac SR vesicles during Ca²⁺ uptake. Five μM tamoxifen (A), 15 μM 4-hydroxytamoxifen (B) or 22 μM clomiphene (C) were added to SR vesicles after Ca²⁺ uptake was started. The traces in (A – C) are superimposed results of control experiments (open symbols) and experiments in which the drugs were added 75 s after uptake started (closed symbols). The drug concentrations used were chosen because they inhibited SR Ca²⁺ uptake to approximately the same extent (based on the results shown in Figure 2).

Figure 4

Measurement of the ATPase activity of the cardiac SR Ca²⁺ pump in the presence of 4-hydroxytamoxifen. (A) NADH concentration as a function of time for a control experiment in which ethanol (final concentration 0.9 μl ml⁻¹) was added to the SR vesicles (downward arrow) 150 s after the experiment started. (B) As in (A) except that 4-hydroxytamoxifen in ethanol (final 4-hydroxytamoxifen concentration 15 μM; final ethanol concentration 0.9 μl ml⁻¹) was added 150 s after the experiment started. The lines in (A) and (B) show the slopes of the curve determined by linear regression from the data points prior to (red lines and red symbols) and after (blue lines and blue symbols) after the addition of ethanol (A) or 4-hydroxytamoxifen (B) to the buffer. For the experiment in (A), the rate of change in [NADH] was 0.182 μM s⁻¹ before the addition of ethanol and 0.201 μM s⁻¹ after the addition; for the experiment in (B), the rate of change of [NADH] was 0.190 μM s⁻¹ before the addition of 4-hydroxytamoxifen and 0.195 μM s⁻¹ after the addition. Sixty μg of SR vesicle protein was used for the experiments in (A) and (B).

Figure 5

Cardiac SR Ca²⁺ uptake in the presence of β-estradiol. (A) V_max for cardiac SR Ca²⁺ uptake in the presence of β-estradiol at concentrations between 1 and 25 μM. Results are expressed as per cent of the mean V_max for control experiments (shown by the open symbol to the left) conducted in the absence of β-estradiol. Uptake was measured with 25 μg SR vesicle protein in the cuvette; the number above each point indicates the number of experiments. (B) β-estradiol (20 μM) had no detectable effect on tamoxifen-dependent inhibition of cardiac SR Ca²⁺ uptake (tamoxifen concentration 5 μM). Although the mean V_max in the presence of β-estradiol was slightly higher than that of the control, this difference was not significant (P=0.086). The numbers above the bars indicate the number of experiments.