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Diethylstilbestrol Is a Potent Inhibitor of Store-Operated Channels and Capacitative Ca²⁺ Influx

Boston University School of Medicine, Boston, Massachusetts (S.I.Z., T.S., F.M., V.M.B.); and Eastern Virginia Medical School, Norfolk, Virginia (Y.D., C.F., P.F.B.)

Received January 22, 2004; accepted June 16, 2004.

	Abstract

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We have recently found that diethylstilbestrol (DES), a synthetic estrogen agonist, inhibits thrombin-induced Ca²⁺ influx in human platelets, but it remains unclear to what extend this effect might be related to the store-operated Ca²⁺ influx pathway. To study the effect of DES on store-operated channels and capacitative Ca²⁺ influx, we used rat basophilic leukemia (RBL) cells, vascular smooth muscle cells (SMC), and human platelets, and recorded whole-cell Ca²⁺ release-activated Ca²⁺ (CRAC) currents and thapsigargin (TG)-induced capacitative Ca²⁺ influx. In this study, we demonstrate that extracellular DES produces a dose-dependent and reversible inhibition of CRAC currents in RBL cells (IC₅₀, 0.5 µM), whereas intracellular DES (25 µM) has no effect. Extracellular DES (up to 30 µM) inhibited only CRAC but did not affect a whole-cell monovalent cation current mediated by TRPM7 channels. DES effectively inhibited TG-induced capacitative Ca²⁺ influx in a dose-dependent manner with an IC₅₀ values of 0.1 µM in RBL cells, <0.1 µM in SMC, and 1 µM in human platelets. It is noteworthy that trans-stilbene, a close structural analog of DES that lacks hydroxyl and ethyl groups, had no effect on CRAC current and on store-operated Ca²⁺ influx. Thus, we found DES to be a very effective inhibitor of store-operated channels and Ca²⁺ influx in a variety of cell types.

We demonstrated recently that some agents that possess a stilbene structural moiety inhibit thrombin-induced Ca²⁺ influx in human platelets (Dobrydneva et al., 1999, 2002), but to what extent this effect could be related to a direct effect on the store-operated channels remained unclear. Among the stilbene analogs, we found diethylstilbestrol (DES) to be the most potent blocker (100% inhibition at 10 µM) (Dobrydneva et al., 2003). trans-Resveratrol, genistein, and tetrahydrochrysenes (rigid analogs of DES) inhibited thrombin-induced Ca²⁺ responses, but their effectiveness was considerably lower (50% inhibition at 10 µM) (Dobrydneva et al., 1999, 2002, 2003).

In this study, we tested and compared the effects of DES and its close derivative, trans-stilbene (trans-S) directly on the CRAC and TRPM7 currents in RBL cells and on the capacitative Ca²⁺ influx triggered by thapsigargin (TG, an inhibitor of sarco(endo)plasmic reticulum Ca²⁺ ATPase) in RBL cells, SMC, and human platelets. As a result of these studies, we found that DES effectively inhibits CRAC but not TRPM7 channels and inhibits capacitative Ca²⁺ influx with IC₅₀ values below 100 nM in a variety of cell types, making DES the most effective inhibitor of CRAC current and capacitative Ca²⁺ influx yet known.

	Materials and Methods

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Cells. Primary culture SMC from rabbit aortas, RBL cells, and human platelets were used in this study as described previously (Dobrydneva and Blackmore, 2001; Trepakova et al., 2001; Smani et al., 2003).

Electrophysiology. Whole-cell currents were recorded in RBL cells using standard whole-cell (dialysis) patch-clamp technique. An Axopatch 200B amplifier (Axon Instruments, Union City, CA) was used; data were digitized at 5 kHz and filtered at 1 kHz. Pipettes were used with tip resistance of 2 to 4 M. After breaking into the cell, holding potential was 0 mV and ramp depolarizations (from –100 to +100mV, 200 ms) were applied every 5 s. The time course of CRAC current development was analyzed at –80 mV in each cell (amplitude was expressed in picoAmperes per picoFarad). The capacitance of RBL cells was 19.6 ± 0.7 pF (n = 19). The maximum current density (in picoAmperes per picoFarad) at –80 mV was determined after 10 min of cell dialysis and summarized for all the cells tested (S.E. is shown in the figures). Representative current-voltage relationships are shown during ramp depolarization after 10 min of cell dialysis. Passive leakage current (0.1–0.5 pA/pF at –80 mV) with zero reversal potential at the moment of breaking into the cell was subtracted. To distinguish CRAC current from the current through TRPM7 channels, 3 mM MgCl₂ was added to the pipette, and 20 mM Ca²⁺ was used from outside when CRAC was recorded. For recording of CRAC current intracellular (pipette) solution contained 145 mM cesium glutamate, 3 mM MgCl₂, 10 mM BAPTA, and 10 mM HEPES, pH 7.2. For registration of TRPM7, MgCl₂ was omitted from the pipette solution. Extracellular solutions were 20 mM CaCl₂, 1 mM MgCl₂, 130 mM NaCl, 3 mM CsCl, and 5 mM HEPES, pH 7.4, for CRAC currents, and 0 Ca²⁺/0 Mg²⁺ with 140 mM NaCl, 3 mM CsCl, 0.1 mM EGTA, 5 mM tetraethylammonium, and 10 mM HEPES, pH 7.4, for TRPM7 currents. For extracellular application, DES (different concentrations) was added to the bath after CRAC or TRPM7 currents reached their maximum. For intracellular application, DES (25 µM) was added to the pipette solution. Experiments were done at 20 to 22°C.

Intracellular Ca²⁺ Measurement. RBL and SMC cells were loaded with fura-2AM, and quantitative changes in intracellular Ca²⁺ (Fura-2, F₃₄₀/F₃₈₀) were monitored as described previously (Trepakova et al., 2001; Smani et al., 2003). For summary data, Ratio was calculated as the difference between the peak ratio after extracellular Ca²⁺ was added, and its level right before Ca²⁺ addition. Summary data are shown after subtraction of the basal Ca²⁺ influx. Dual-excitation fluorescence imaging system (IonOptics) was used for studies of individual SMC and RBL cells. Data were summarized from the large number of individual SMC or RBL cells (from three to five different preparations). The basal Ca²⁺ influx was 0.3 ± 0.1 (n = 44) in RBL cells and 0.7 ± 0.1 (n = 22) in SMC and was subtracted from summary data but not from the original traces.

In human platelets, Ca²⁺ was measured using Fura-2 technique as described previously (Dobrydneva and Blackmore, 2001). In brief, platelets were isolated from citrated blood by centrifugation and incubated in modified Tyrode's buffer containing 1 mM EGTA. Platelets were incubated for 1 h with 2 µM Fura-2 acetoxymethyl ester, washed, and resuspended in Tyrode's buffer without calcium. Calcium measurements were performed on platelets in suspension using a SPEX ARCM spectrofluorometer.

Drugs. All the salts and drugs were from Sigma (St. Louis, MO). Statistical Analysis. Summary data are presented as mean ± S.E. A logistic function was used to fit the dose-dependence. Student's t test was used to determine the statistical significance of the obtained data. The significance between multiple groups was evaluated using analysis of variance. Data were considered significant at P < 0.01 (*).

	Results and Discussion

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Dose-Dependent Inhibition of CRAC Currents by DES. Fig. 1A shows a typical example of the time course of development of the whole-cell CRAC current (at –80 mV) in RBL cells in the presence of 20 mM extracellular Ca²⁺ when the cells are dialyzed with 10 mM BAPTA (which is commonly used to deplete their Ca²⁺ stores). Application of the increasing doses of DES (1 and 10 µM) to RBL cells caused fast inhibition of CRAC current. Fig.1B presents current voltage relationships of CRAC currents in the presence of DES (1 and 10 µM). The dose dependence of DES-induced inhibition of CRAC current (Fig. 1C) shows that DES started to suppress CRAC at concentrations as low as 100 nM and totally inhibited CRAC when 10 µM was applied. It is noteworthy that the effect of DES was monophasic, and (contrary to 2-APB) it did not cause any activation of CRAC currents or Ca²⁺ influx at low concentrations. IC₅₀ for DES-induced inhibition of CRAC currents was around 0.6 µM, making DES the most potent inhibitor of CRAC current known so far. We found the effect of DES to be reversible even when used at high concentrations: after application of 3 µM of DES, the amplitude of CRAC current was restored to 92 ± 2% of its original value after 12 to 15 min of washing (n = 4). Even after 30 µM DES was applied, 15 min of washing was enough to restore CRAC current to 82 ± 2% of its original value. To get a better understanding of the mechanism of DES action, we also tested its effects from inside the cell. Fig.2 shows that the dialysis of RBL cells with DES (even at a very high concentration of 25 µM) did not affect the development, or the amplitude of CRAC current. Thus, we found that DES is inhibiting CRAC channels only when applied from outside the cell, which suggests that it is most probably a direct inhibitor of store-operated channels working from the outside that does not involve intracellular signaling targets or pathways.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Whole-cell CRAC current is inhibited by extracellular DES in RBL cells. A, representative time course of the whole-cell inward CRAC current that develops during cell dialysis with BAPTA-containing solution in control extracellular solution that contains 20 mM Ca2+. DES (1 and 10 µM) was applied to the external solution at the times specified by the bars. The current density (in picoAmperes per picoFarad) is shown at –80 mV. Zero time corresponds to the beginning of cell dialysis. Passive leakage current (at the moment of breaking into the cell) was subtracted. B, typical example of the current-voltage relationships of CRAC current before (1) and after 1 µM (2) and 10 µM (3) DES was applied to the cell in the experiment shown in A. C, dose-dependence of the effects of DES and trans-S on CRAC current. Summary data (with S.E.), with each concentration tested in the number of cells (as specified by the numbers). D, the chemical structures of trans-S and DES are shown on the right.

View larger version (10K): [in this window] [in a new window]   Fig. 2. CRAC current is not affected by intracellular application of DES. A, summary data showing the development of the whole-cell inward CRAC current in RBL cell dialysis with intracellular solution containing BAPTA (10 mM) with or without DES (25 µM). , the average (± S.E.) current density (at –80 mV) in control cells (n = 11); , current density (at –80 mV) in the cells dialyzed with DES (n = 11). Zero time corresponds to the beginning of cell dialysis. B, average current-voltage relationships (± S.E.) of maximum CRAC current in control RBL cells () and in cells dialyzed with DES (25 µM) in the experiments shown in A.

Monovalent Cation Currents That Are Mediated by TRPM7 Channels Are Not Affected by DES. It is known that in the absence of extracellular divalent cations, CRAC channels start to conduct monovalent cations, and monovalent cation current through CRAC channels could be observed in RBL cells in divalent cation-free (DCF) conditions. However, it is now well accepted that not only CRAC but also TRPM7 channels [called also MagNuM (Nadler et al., 2001), MIC (Prakriya and Lewis, 2002), or MCC (Mubagwa et al., 1997; Zakharov et al., 2003)] are activated in RBL and other cells under DCF conditions (Bakowski and Parekh, 2002a; Hermosura et al., 2002; Prakriya and Lewis, 2002), and discrimination of these two currents poses a certain difficulty. 2-APB, which was originally thought and used as a specific inhibitor of CRAC channels, was found to also inhibit TRPM7 channels in almost the same range of concentrations (Bakowski and Parekh, 2002b; Prakriya and Lewis, 2002; Zakharov et al., 2003; Kozak and Cahalan, 2003). To determine whether the inhibitory effects of DES are specific to CRAC channels, we tested the effects of DES (up to 30 µM) on the monovalent cation currents that are mediated by TRPM7 channels. Fig. 3 shows a typical example of the development of the monovalent cation current (in the absence of extracellular divalents) through TRPM7 channels. Corresponding current voltage relationships are shown in Fig. 3B. Application of 20 µM DES (which totally inhibited CRAC current) produced no effect on TRPM7 current (n = 6), which could be subsequently inhibited by spermine (100 µM). We have recently shown that spermine specifically inhibits inward component of the monovalent current mediated by TRPM7 but not CRAC channels (Zakharov et al., 2003). Thus, DES seemed to be a potent inhibitor of CRAC but not TRPM7 channels and could be used as a new tool to effectively separate these two channels.

View larger version (13K): [in this window] [in a new window]   Fig. 3. DES does not affect whole-cell current through TRPM7 channels. A, representative time course of the whole-cell current that develops in divalent cation-free conditions (intracellular dialysis with 10 mM BAPTA and 0 Mg2+, and extracellular 0 Ca2+/0 Mg2+). DES (20 µM) applied to the cell did not affect the current, whereas spermine (SpM; 10 µM) inhibited it. The current density (in picoAmperes per picoFarad) is shown at –80 mV. Zero time corresponds to the beginning of cell dialysis. Passive leakage current (at the moment of breaking into the cell) was not subtracted. B, typical example of the current-voltage relationships of TRPM7 current in the absence (1) and in the presence of 20 µM DES (2) and 100 µM spermine (3) DES was applied to the cell in the experiment shown in A.

The Effects of DES and Its Structural Analog on Capacitative Ca²⁺ Influx in RBL Cells, SMC, and Platelets. Fig. 4A shows that 1 µM DES inhibited 93 ± 3% of the capacitative Ca²⁺ influx triggered by TG-induced depletion of Ca²⁺ stores in RBL cells. The inhibitory effect of DES was dose-dependent with IC₅₀ 0.1 µM (Fig. 4B), which was even lower than for the CRAC currents. Thus, DES indeed seemed to be very potent in inhibiting both CRAC currents and capacitative Ca²⁺ influx in RBL cells.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Comparative analysis of the effects of DES and trans-S on capacitative Ca2+ influx in RBL cells, SMC, and human platelets. Intracellular Ca2+ (fura-2 ratio) was monitored in RBL cells, SMC, and human platelets after TG was added in the absence of extracellular Ca2+ to deplete their stores (2 µM for RBL and SMC; 0.1 µM for platelets). A, C, and E, representative traces showing the results of addition of extracellular Ca2+ (2 mM, 5 min after addition of TG) in control cells (TG) and when DES (1 µM) or trans-S (10 µM) was added 1 min before Ca2+ (at the time shown by the asterisk). In platelets, 10 µM DES was used. B, D, and F, dose dependence of the effects of DES and trans-S on TG-induced Ca2+ influx in RBL cells, SMC, and platelets. Summary data are shown for the number of SMC and RBL cells specified by the numbers above each data point (from three different cultures). For platelets, the data are summarized from five experiments. Basal Ca2+ influx is subtracted.

Next, we tested whether DES effects were specific to Ca²⁺ selective CRAC channels, or if it could also inhibit store-operated nonselective cation channels, which are known to mediate capacitative Ca²⁺ influx in SMC and platelets. Figure 4, C and D, shows that indeed, capacitative Ca²⁺ influx in SMC is inhibited by DES in a dose-dependent manner with IC₅₀ < 0.1 µM. In platelets, the same effect was observed (Fig. 4, E and F). Thus, DES was effective in inhibiting capacitative Ca²⁺ influx independently on whether it is mediated by CRAC (in RBL cells, Fig. 4, A and B), or by SOC (in SMC, Fig. 4, C and D; in platelets, Fig. 4, E and F) channels.

It is noteworthy that only DES, and not its very close structural analog trans-stilbene, was able to inhibit capacitative Ca²⁺ influx in RBL cells, SMC, and human platelets (Fig. 4). trans-Stilbene also had no effect on CRAC currents in RBL cells (Fig. 1C).

Summarizing our findings, we would like to propose DES as a new and very potent inhibitor of store-operated channels and capacitative Ca²⁺ influx in a variety of cell types. It could be a prototype drug to develop new potent and truly specific inhibitors of store-operated channels. It could be also used as a helpful tool in discriminating CRAC and TRPM7 channels. The molecular target of DES action on CRAC is unknown, but because DES inhibits store-operated channels only when applied from the outside, the effects are rapid and easily reversible, they most likely do not involve any intracellular targets or transcription/translation processes, and they may be localized to the channel itself or to some extracellularly located and closely related regulatory proteins. We may speculate that because DES is known to bind to the estrogen receptor, it is possible that the binding site for DES on the channel (or regulatory proteins) may share some homology with the binding site on the estrogen receptor.

	Footnotes

 
The laboratory of V.M.B. was supported by National Institutes of Health grant HL54150. T.S. was supported by American Heart Association grant 0225688T. The laboratory of P.F.B. was supported by grants from the Jeffress Memorial Trust and the Commonwealth Health Research Board. Y.D. was supported by American Association of University Women Educational Foundation and by the United States Department of Agriculture.

S.I.Z. and T.S. contributed equally to this work

ABBREVIATIONS:SOC, store-operated Ca²⁺-conducting; CRAC, Ca²⁺ release-activated Ca²⁺-selective; SMC, smooth muscle cells; 2-APB, 2-aminoethoxydiphenyl borate; DCF, divalent cation-free; DES, diethylstilbestrol; trans-S, trans-stilbene; TG, thapsigargin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; DCF, divalent cation free.

Address correspondence to: Dr. Victoria M. Bolotina, Vascular Biology Unit, EBRC, X-704, Boston, Massachusetts, USA. E-mail: bolotina{at}bu.edu

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