Introduction

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2-Aminoethoxydiphenyl borate (2APB) was originally characterized as a cell-permeable inhibitor of inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (Maruyama et al., 1997). 2APB inhibited IP₃-induced Ca²⁺ release from cerebellar microsomes without affecting IP₃ binding. 2APB also inhibited agonist-induced increases in intracellular free calcium ([Ca²⁺]_i) in platelets and neutrophils and blocked agonist-induced contractions in thoracic aorta, but it had no effect on KCl-induced contractions. 2APB has been used extensively to inhibit the release of intracellular Ca²⁺ (Cui and Kanno, 1997; Ascher-Landsberg et al., 1999; Gysembergh et al., 1999; Hamada et al., 1999; Ma et al., 2000; van Rossum et al., 2000).

2APB has the ability to form a five-membered boroxazolidine heterocyclic ring (Fig. 1) when an internal coordinate bond is formed between the nitrogen in the ethanolamine side chain and the tricoordinated boron (Strang et al., 1989). This heterocyclic form of 2APB (B,B-diphenylboroaxozolidine) forms crystals in staggered arrays of molecules. Each molecule links with two others through hydrogen bonds (Rettig and Trotter, 1976); this feature most probably accounts for the fact that 2APB is soluble in water (see below). This heterocyclic species of 2APB would be more hydrophobic than the compound without the heterocyclic ring and should enter cells more rapidly than the primary amine open-chain species that could be protonated. It is also known that boron-nitrogen coordination results in the formation of dimers (Nöth, 1970); van Rossum et al. (2000) suggested that 2APB also exists as a dimer (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Structures of 2APB dimer, 2APB monomer ring, 2APB monomer, xestospongin C, 2,2-diphenyltetrahydrofuran, diphenylboronic acid, phenytoin, and diphenhydramine. The 2APB monomer ring is formed when the boron forms a coordinate bond with the nitrogen, giving boron a full outer-shell octet of electrons, thus allowing for a tetragonal boron bond configuration (Strang et al., 1989). This compound will not carry a positive charge, whereas the 2APB monomer (no ring) will probably have a positive charge at physiological pH because it is a primary amine. Considering the ability of the boron in 2APB to form a coordinate bond with the ethanolamine nitrogen, neutral polymers of 2APB could theoretically exist, such as the 2APB dimer shown.

The recent study conducted by Ma et al. (2000) relied on the specificity of 2APB as a blocker of Ca²⁺ release via the IP₃ receptor in the endoplasmic reticulum (ER) of several different cell lines. The authors discounted a direct effect of 2APB on plasma membrane Ca²⁺ channels (see Fig. 4 in Ma et al., 2000). In another study, van Rossum et al. (2000) showed that 2APB, when added with thapsigargin to DDT₁-MF2 cells had no effect on the Ca²⁺ release component but inhibited the Ca²⁺ entry component (see Fig. 1 in van Rossum et al., 2000). An interpretation of this result, although not considered by the authors, was that 2APB was blocking SOCC directly.

We have been investigating the mechanism by which phytoestrogens were able to inhibit platelet aggregation and found that several phytoestrogens inhibited Ca²⁺ influx in platelets induced by thrombin (Dobrydneva et al., 1999). The phytoestrogens were inhibiting the entry of Ca²⁺ through SOCC, because the phytoestrogen trans-resveratrol was also able to inhibit thapsigargin-mediated Ca²⁺ influx and basal Ba²⁺ ion influx (Dobrydneva et al., 1999). Thapsigargin is believed to promote Ca²⁺ entry through SOCC by depleting the IP₃-sensitive Ca²⁺ stores in platelets (Sage, 1997). To further investigate the mechanism by which phytoestrogens inhibit Ca²⁺ influx, we sought to prevent thrombin-mediated Ca²⁺ mobilization from IP₃-sensitive Ca²⁺ stores by using 2APB.

The mechanism by which SOCC is activated in a variety of cells has received much attention recently (Putney, 1999a,b; Berridge et al., 2000). Current evidence supports a model in which there is reversible trafficking and coupling of the IP₃ receptor/channel with the plasma membrane SOCC, a process called conformational coupling (Kiselyov et al., 1998; Boulay et al., 1999; Patterson et al., 1999; Yao et al., 1999; Ma et al., 2000). In platelets, evidence supports this conformational coupling mechanism because coimmunoprecipitation experiments show a coupling of endogenously expressed hTrp1 with type II IP₃ receptors when intracellular Ca²⁺ stores are depleted (Rosado et al., 2000; Rosado and Sage, 2000a). There is some very strong evidence against the conformational coupling mechanism for SOCC activation. When the IP₃ receptor-deficient B-cell line DT40 was stimulated with anti-IgM or carbachol, there was no increase in [Ca²⁺]_i; however, the ability of thapsigargin to increase [Ca²⁺]_i was unaffected (Sugawara et al., 1997). These data were interpreted to mean that IP₃ receptors were not the mediator between the endoplasmic reticulum and SOCC. Also, studies in T lymphocytes lacking type 1 IP₃ receptors showed that depletion of intracellular stores with thapsigargin resulted in stimulation of Ca²⁺ influx, whereas agonist-induced influx was inhibited (Jayaraman et al., 1995).

In the study by Maruyama et al. (1997), 2APB was shown to inhibit thrombin-mediated elevation in [Ca²⁺]_i when extracellular Ca²⁺ was present. However, the effect of 2APB to inhibit thrombin-mediated intracellular Ca²⁺ mobilization (platelets incubated without extracellular Ca²⁺, which prevents Ca²⁺ influx) was not examined (Maruyama et al., 1997). Also, the effect of 2APB on SOCC activation by thapsigargin was not investigated in platelets (Maruyama et al., 1997). In the present study, 2APB inhibited thrombin-induced intracellular Ca²⁺ mobilization in platelets but not thapsigargin-mediated intracellular Ca²⁺ mobilization. However, thapsigargin-stimulated Ca²⁺ influx after store depletion was inhibited by 2APB. 2APB also rapidly inhibited basal uptake of Sr²⁺ and Mn²⁺, which suggests a direct effect of 2APB on plasma membrane SOCC channels. Therefore, 2APB may not be a specific inhibitor of IP₃-mediated intracellular Ca²⁺ mobilization as currently believed. Nevertheless, 2APB rapidly inhibited thrombin- and thapsigargin-mediated increases in [Ca²⁺]_i in human platelets by at least two different mechanisms.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Reagents and Material Sources. Thrombin and EGTA were obtained from Sigma Chemical Co. (St. Louis, MO). Thapsigargin, cyclopiazonic acid, and ionomycin were obtained from Calbiochem (San Diego, CA). Fura-2/AM was from Molecular Probes (Eugene, OR). Diphenylboronic acid, ethanolamine ester [also called 2-aminoethoxydiphenyl borate (CAS registry number [524-95-8])], diphenylboronic anhydride, and 2,2-diphenyltetrahydrofuran were from Aldrich Chemical (Milwaukee, WI). Other chemicals and reagents were from Fisher Scientific (Fair Lawn, NJ) and Sigma.

Blood Donors and Platelet Preparation. All donors were healthy volunteers (aged 20-40 years) who had not consumed any medication known to affect platelet function (e.g., calcium-channel blockers and aspirin) for at least 10 days before the study. Venous blood was collected into 1/10 volume of 74.8 mM sodium citrate, 38.1 mM citric acid, and 123 mM dextrose, pH 6.4 (Baxter, McGaw Park, IL). The blood was centrifuged at 250g for 10 min at room temperature to obtain platelet-rich plasma. The platelet-rich plasma was centrifuged at 550g for 12 min to sediment the platelets. The platelets were then suspended in a modified Tyrode's physiological salt solution (e.g., 145 mM NaCl, 4 mM KCl, 1 mM MgSO₄, 0.5 mM Na₂HPO₄, 10 mM Na/HEPES, and 6 mM glucose; pH 7.4) containing 1.0 mM EGTA, which acted to prevent spontaneous aggregation during the various experimental manipulations by binding extracellular Ca²⁺ (Nolan and Lapetina, 1990). The platelets were washed once (at 500g for 15 min) and finally suspended Tyrode's solution, nominally Ca²⁺-free (without EGTA), at a count of approximately 3 × 10⁸ platelets/ml. In experiments involving the use of La³⁺, the platelets were suspended in Tyrode's solution without Na₂HPO₄ to prevent the formation of insoluble lanthanum phosphate.

Drug Solution Preparations. Stock solutions of the drugs (thapsigargin, 2APB, diphenylboronic anhydride, and 2,2-diphenyltetrahydrofuran) in Me₂SO (10 mM) were prepared and stored at 20°C. Just before each experiment, aliquots were thawed and diluted to the desirable concentration with Me₂SO (see individual figure legends for concentrations used). In some experiments, we dissolved 2APB in water (10 mM), although vigorous shaking of the suspension was required for it to go into solution. This aqueous solution of 2APB produced the same results as 2APB dissolved in Me₂SO or ethanol (data not shown).

Platelet Loading with Fura-2 and Measurement of [Ca²⁺]_i. Calcium measurements [Ca²⁺]_i were made using the fluorescent dye fura-2, which involved incubating the platelets with the cell-permeating acetoxymethyl ester (fura-2/AM) (Sargeant et al., 1992). A suspension of human platelets (isolated as described above) was incubated with 2 µM fura-2/AM for 1 h at room temperature on a rocking platform. Excess fura-2/AM was removed by centrifugation (500g for 10 min), and the platelets were suspended in Tyrode's solution without added Ca²⁺ or EGTA. Platelet suspensions (0.5 ml) were placed into 1.5-ml aggregometer tubes containing a magnetic stir bar (CHRONO-LOG, Havertown, PA). Just before [Ca²⁺]_i measurements were performed, Ca²⁺ was added back to the platelets to a final concentration of 1.0 mM 30 s before the commencement of the experiment, then 2APB (various concentrations), thapsigargin, or thrombin was added (see individual figure legends for concentrations used). The various agents were added to the cuvette as data was being collected; if the pipette tip was placed into the light path, a small transient decrease in fluorescence ratio was observed. The measurements of [Ca²⁺]_i was performed at room temperature in a SPEX ARCM spectrofluorometer (SPEX Industries, Edison, NJ) using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Calibration was performed as described previously for human sperm (Blackmore et al., 1990). [Ca²⁺]_i was calculated with the use of the SPEX dM3000 software package.

Measurement of [Ba²⁺]_i and [Sr²⁺]_i. To assess basal Ca²⁺-channel activity without agonist stimulation, Sr²⁺ or Ba²⁺ was added to platelets in Ca²⁺-free medium to act as a Ca²⁺ surrogate. Sr²⁺ and Ba²⁺ enter the cell through Ca²⁺ channels; unlike Ca²⁺, however, Ba²⁺ cannot be extruded from the cell by plasma membrane Ca²⁺-ATPase pump, whereas Sr²⁺ can be extruded (Ozaki et al., 1992). Once inside the cell, Sr²⁺ and Ba²⁺ form fluorescent complexes with the dye fura-2 in a manner similar to that of Ca²⁺ but with a different affinity. The intensity of the fluorescence is directly proportional to the [Sr²⁺]_i or [Ba²⁺]_i. SrCl₂ or BaCl₂ (10 mM) was added to the fura-2-loaded platelets in the absence of extracellular Ca²⁺ and in the absence of agonist. The fura-2/Ba²⁺ or fura-2/Sr²⁺ fluorescent complex was measured, and the results were expressed as 340:380-nm ratios. To show that Ba²⁺ and Sr²⁺ were entering through Ca²⁺ channels, Ca²⁺ (1.0, 2.0, 3.0, and 5.0 mM) was added to the platelets along with either Sr²⁺ or Ba²⁺. The presence of Ca²⁺ acted as a competitor of Sr²⁺ and Ba²⁺ influx because Ca²⁺ produced a dose-dependent reduction of the 340/380-nm fluorescence signal that was increased by Sr²⁺ and Ba²⁺ influx (data not shown).

Thapsigargin-Induced [Ca²⁺]_i Entry. After Ca²⁺ was added back to the platelet suspensions, thapsigargin (dissolved in Me₂SO) was added to a final concentration of 100 nM, and the fluorescence was monitored as described previously (Dobrydneva et al., 1999). Alternatively, thapsigargin was added to platelets in the absence of extracellular Ca²⁺ after several minutes of incubation with EGTA (see specific experiments). Ca²⁺ was then added to activated platelets, and this resulted in a rapid increase in [Ca²⁺]_i, which predominantly represented Ca²⁺ influx through SOCC. The same results were obtained if either 0.1 mM EGTA was added or no EGTA was used (Tyrode's solution used was nominally Ca²⁺-free because no Ca²⁺ was added).

Molecular Modeling. Molecular modeling and energy minimization protocols were performed using CambridgeSoft Chem3D software (version 3.5.1; Cambridge, MA). Minimal energy conformations were obtained using the default settings provided in the MM2 (molecular mechanics) calculation package, part of the Chem3D software.

Statistical Analysis. Data are reported as mean ± S.E.M. for the number of individual experiments specified in each figure legend. Different platelet donors were used for each experiment.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Effect of 2APB on Thrombin and Thapsigargin to Increase [Ca²⁺]_i in the Presence and Absence of Extracellular Ca²⁺. The data in Fig. 2A show the effect of three different concentrations of 2APB on the action of 0.05 U/ml thrombin to increase [Ca²⁺]_i. The 2APB was added approximately 30 s before thrombin, and subsequent experiments (see below) show that this preincubation was not required to observe 2APB-mediated inhibition of agonist-induced elevation of [Ca²⁺]_i. A concentration of 100 µM 2APB produced total inhibition of thrombin and 10 µM 2APB caused an approximately 50% inhibition, whereas 1 µM 2APB produced a small suppression in the peak effect and slowed the rate of [Ca²⁺]_i increase. The addition of 1.0 mM EGTA to the medium reduced the ability of thrombin to increase [Ca²⁺]_i substantially, the increase in [Ca²⁺]_i being approximately 30 nM (Fig. 2B), whereas when extracellular Ca²⁺ was present, the increase was approximately 240 nM (Fig. 2A). 2APB inhibited, in a dose-dependent manner, the ability of thrombin to increase [Ca²⁺]_i in the absence of extracellular Ca²⁺ and, hence, in the absence of Ca²⁺ influx. These results therefore confirm the findings of Maruyama et al. (1997) by showing that 2APB was able to inhibit the ability of thrombin to increase [Ca²⁺]_i in human platelets when extracellular Ca²⁺ was present. In addition, we also demonstrate that 2APB inhibited thrombin-mediated mobilization of intracellular Ca²⁺ when extracellular Ca²⁺ was absent (Fig. 2B). Therefore, 2APB was able to inhibit both intracellular Ca²⁺ mobilization and Ca²⁺ influx, either directly on the Ca²⁺ influx channel or indirectly by its capacity to inhibit IP₃-mediated Ca²⁺ store depletion and, hence, prevent activation of SOCC by conformational coupling. The data in Fig. 3 summarize the dose-response data for 2APB to inhibit the ability of thrombin to increase [Ca²⁺]_i when extracellular Ca²⁺ was present (Fig. 2A) and absent (Fig. 2B). Although not shown, 1, 10, and 100 µM 2APB had no effect on basal Ca²⁺]_i. The addition of 500 µM of 2APB produced an elevation in [Ca²⁺]_i that was the same whether extracellular calcium was present or not. This suggests that the increase in [Ca²⁺]_i was caused by intracellular mobilization of Ca²⁺ by 2APB; this Ca²⁺-mobilizing effect was not examined any further in the present study.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of 2APB on thrombin-mediated increase in [Ca2+]i in the presence (A) and absence (B) of extracellular Ca2+. Platelets were loaded with fura-2 as described under Materials and Methods and incubated in the absence of added extracellular Ca2+, which was nominally Ca2+-free. Calcium (1.0 mM) was added to 0.5 ml of platelets 30 s before data collection was started. Various concentrations of 2APB in Me2SO were added to the platelets at 5 s, and thrombin was added at 30 s. A representative of five experiments is shown (A), and the averages of the inhibitory effects of 2APB from these experiments are shown in Fig. 3. B, platelets were incubated in the absence of added Ca2+, and 1.0 mM EGTA was added to the platelets 30 s before data collection was started. After 5 s, 100 µM 2APB or Me2SO (control) was added; 20 s later thrombin was added. The increase in [Ca2+]i observed under these conditions was much smaller than the increase in [Ca2+]i observed when Ca2+ was present in the medium (A). 2APB was able to produce a large inhibition of the increase in [Ca2+]i elicited by thrombin. A representative of four experiments is shown, and the average of these experiments are shown in Fig. 3.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Dose response of 2APB to inhibit 0.05 U/ml thrombin (with and without extracellular Ca2+) and 0.1 µM thapsigargin-mediated increases in [Ca2+]i (with extracellular Ca2+). The inhibitory effect of 2APB on basal Sr2+ influx is also shown. The data presented are from experiments shown in Figs. 2, 4, and 6A. The values are means ± S.E.M. from four to five separate experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Dose response of 2APB to inhibit thapsigargin-mediated (0.1 µM) increases in [Ca2+]i when extracellular Ca2+ was present. Calcium (1.0 mM) was added to the platelets 30 s before data collection was started. 2APB was added at 10 s, and 0.1 µM thapsigargin was added at 30 s. A representative of five experiments is shown. The average of the five experiments is shown in Fig. 3.

Measurement of Ca²⁺ Influx Using the Ca²⁺ Surrogates Sr²⁺, Ba²⁺, and Mn²⁺. The data in Fig. 5 show the effect of adding Ba²⁺ directly to platelets in the absence of Ca²⁺ and without any agonist. When Ba²⁺ enters the fura-2-loaded platelet, it binds to fura-2 and causes an increase in the 340/380 nm fluorescence ratio (Dobrydneva et al., 1999). After a slight delay, Ba²⁺ caused an increase in the 340/380 nm fluorescence ratio, and 2APB displayed a dose-dependent inhibition of Ba²⁺ influx, consistent with 2APB directly inhibiting the influx of Ba²⁺ through plasma membrane Ca²⁺ channels. We showed previously that thapsigargin potentiated Ba²⁺ influx; therefore, Ba²⁺ influx represents, at least in part, the activity of SOCC (Dobrydneva et al., 1999). Likewise, when Sr²⁺ was added to fura-2-loaded platelets in the absence of extracellular Ca²⁺ and agonist, 2APB caused a dose-dependent inhibition of Sr²⁺ influx (Fig. 6A). The data showing the dose-dependent effect of 2APB to inhibit Sr²⁺ influx is summarized in Fig. 3. We believe that Sr²⁺ influx (Fig. 6A) occurs predominantly via SOCC because thapsigargin was able to stimulate Sr²⁺ influx further (Fig. 6B). When thapsigargin was added to platelets in the absence of extracellular Ca²⁺, there was a very small increase in [Ca²⁺]_i (approximately 5% increase over basal within several minutes) (also see Figs. 9B and 10B). This result indicates that the increase in 340/380 nm fluorescence ratio (Figs. 5 and 6) was caused by an influx of Ba²⁺ or Sr²⁺, not by Ca²⁺ release from the ER, because this could only represent a very small contribution to the overall fura-2 signal when extracellular Ca²⁺ was absent (Rosado and Sage, 2000c). The thapsigargin-stimulated Sr²⁺ influx was also inhibited by 2APB in a dose-dependent manner (Fig. 6B), with 100 µM 2APB causing complete inhibition of Sr²⁺ influx. These results are also compatible with the inhibition of SOCC activity by 2APB.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Dose response of 2APB to inhibit Ba2+ influx in unstimulated platelets (no agonist) in the absence of extracellular Ca2+. Platelets were incubated in the absence of added extracellular Ca2+. At 5 s, 2APB or Me2SO solvent control was added. At 30 s, 10 mM BaCl2 was added, and the 340/380 nm fluorescence ratio was measured. 2APB produced a dose-dependent inhibition of Ba2+ influx as measured by a decrease in the fura-2 fluorescence ratio. This result implies that 2APB was blocking Ca2+ channels directly. A representative of four experiments is shown.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Dose response of 2APB to inhibit Sr2+ influx into unstimulated platelets, without extracellular Ca2+ (A), and influence of thapsigargin on Sr2+ influx and the effect of 2APB on thapsigargin-stimulated Sr2+ influx (B). Platelets were incubated in the absence of added extracellular Ca2+. Various concentrations of 2APB were added 10 s before data collection was started. At 5 s, 10 mM SrCl2 was added. The increase in fura-2 340/380 nm fluorescence ratio indicates the entry of Sr2+ through Ca2+ channels. 2APB blocked the entry of Sr2+, suggesting a direct effect on the plasma membrane Ca2+ channels (A). A representative of four experiments is shown. B, platelets were incubated in the absence of extracellular Ca2+. Various concentrations of 2APB were added 10 s before data collection was initiated. Thapsigargin (100 nM) was added at 10 s, and 10 mM SrCl2 was then added at 20 s. Thapsigargin treatment greatly potentiated the influx of Sr2+, such that Sr2+ influx increased without delay. This result suggests that Sr2+ influx in platelets predominantly represents SOCC because thapsigargin was able to stimulate Sr2+ influx. The ability of thapsigargin to increase Sr2+ influx was inhibited by 2APB, with 100 µM 2APB causing an almost total inhibition of thapsigargin-stimulated Sr2+ influx. A representative of four experiments is shown.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Dose response of 2APB to inhibit thrombin-mediated Sr2+ influx into platelets. Platelets were incubated in the absence of added extracellular Ca2+. Thrombin 0.02 U/ml was added at 15 s, and at 200 s, 10 mM SrCl2 was added together with various concentrations of 2APB. 2APB produced an immediate dose-dependent inhibition of Sr2+ influx as measured by a decrease in the fura-2 fluorescence ratio compared with the control. A representative of four experiments is shown.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   The effect of 2APB on Mn2+ influx into platelets as measured by quenching of fura-2. Platelets were incubated in the absence of added extracellular Ca2+. 2APB or Me2SO was added at 5 s, and at 20 s, 1.0 mM Mn2+ was added. There was a slight decline in fura-2 fluorescence with 2APB or Me2SO (without Mn2+), and the addition of Mn2+ produced a rapid decline in fura-2 fluorescence measured at 360 nm. The presence of 2APB was able to almost totally prevent the Mn2+-induced quenching of fura-2, such that the rate of fura-2 fluorescence decline was the same as that for the Me2SO control. A representative of three experiments is shown.

Measurement of SOCC Activity after Depletion of Intracellular Ca²⁺ Stores with Thrombin, Thapsigargin, or Thrombin Plus Thapsigargin. Another common approach used to examine SOCC activity is to mobilize intracellular Ca²⁺ using an agonist (e.g., thrombin) or thapsigargin in the absence of extracellular Ca²⁺ plus EGTA in the medium (Fig. 7). In platelets, this results in a small increase in [Ca²⁺]_i caused by the mobilization of Ca²⁺ from intracellular pools when agonists are added (Rosado et al., 2000). To observe the SOCC activity, Ca²⁺ is then added back to the stimulated cells. A large and rapid influx of Ca²⁺ is then observed (Putney and McKay, 1999). The data in Fig. 9A show the effect of thrombin to mobilize intracellular Ca²⁺ in the absence of extracellular Ca²⁺. A small increase in [Ca²⁺]_i was seen that declined gradually as intracellular Ca²⁺ pools were being depleted and Ca²⁺ was extruded from the cell by the plasma membrane Ca²⁺ ATPase (Paszty et al., 1998). At 150 s, 2.0 mM Ca²⁺ was added to the platelets in the presence and absence of 2APB (2APB and Ca²⁺ were added simultaneously). The presence of 2APB almost totally inhibited the increase in [Ca²⁺]_i. The addition of 2APB alone to thrombin-treated platelets in the absence of extracellular Ca²⁺ caused a gradual decline in [Ca²⁺]_i to basal levels within approximately 2 min (data not shown). This decrease in [Ca²⁺]_i, in the absence of extracellular Ca²⁺, would be consistent with either Ca²⁺ being sequestered again into the ER by SERCA and/or being expelled from the cell by the Ca²⁺-ATPase pump (Paszty et al., 1998) after 2APB inhibition of Ca²⁺ release from the ER by the IP₃ receptor (Maruyama et al., 1997).

View larger version (38K): [in this window] [in a new window]   Fig. 9.   The effect of 2APB on calcium influx initiated after mobilization of intracellular Ca2+ by thrombin (A), thapsigargin (B and C), and thrombin plus thapsigargin (D). A, platelets were incubated in the absence of extracellular Ca2+ and in the presence of 1.0 mM EGTA. Thrombin was added at 10 s. At 150 s, when [Ca2+]i was declining because of depletion of intracellular stores, 2.0 mM Ca2+ with or without 2APB was added simultaneously to the platelets. In the presence of 100 µM 2APB, there was almost no increase in [Ca2+]i. A representative of three experiments is shown. B, platelets were incubated in the absence of extracellular Ca2+. Thapsigargin (100 nM) was added at 20 s, and [Ca2+]i began to increase gradually, leveling off between 10 and 15 min. At 15 min, 1.0 mM Ca2+ with or without 2APB was added simultaneously to the platelets. The presence of 100 µM 2APB totally prevented the increase in [Ca2+]i that was observed in the absence of 2APB when Ca2+ was added. A representative of three experiments is shown. C, the effect of 2APB on the ability of thapsigargin to increase [Ca2+]i was examined in the absence of extracellular Ca2+. 2APB had no effect on thapsigargin to increase [Ca2+]i. A representative of three experiments is shown. D, platelets were incubated in the absence of Ca2+ and with 1.0 mM EGTA. Thapsigargin (0.1 µM) plus thrombin (0.01 U/ml) were added at 5.0 and 10.0 s, respectively. At 250 s, either 2APB alone, 2APB plus Ca2+, or Ca2+ alone was added. 2APB completely prevented the increase in [Ca2+]i induced by the addition of Ca2+ to the extracellular fluid. 2APB alone had a small effect on [Ca2+]i stimulated by thapsigargin and thrombin. A representative of three experiments is shown.

View larger version (33K): [in this window] [in a new window]   Fig. 10.   Time course for the effect of La3+ and 2APB to inhibit the increase in [Ca2+]i elicited by thrombin (A) and thapsigargin (B) in the presence of extracellular Ca2+. C, the effect of EGTA, 2APB, and 2APB plus EGTA to inhibit thrombin-mediated Ca2+ influx is shown. A, thrombin was added to platelets at 10 s in the presence of 1.0 mM extracellular Ca2+. At 60 s, either La3+, 2APB, or both were added. The [Ca2+]i began to decline immediately (A). The inhibitory effects of La3+ and 2APB were not additive, Also, the inhibitory effects of 2APB and La3+ were the same, which suggests that both agents were inhibiting the same Ca2+ channel(s) in platelets. A representative of three experiments is shown. B, thapsigargin (100 nM) was added to platelets at 10 s in the absence of added Ca2+. The influx of Ca2+ was then initiated by adding 1.0 mM Ca2+ at 130 s. When the increase in [Ca2+]i had reached a maximal level, either La3+, 2APB, both were added. Both La3+ and 2APB caused an immediate decline in [Ca2+]I, and the inhibitory effect observed when both La3+ plus 2APB were added together was no greater than that seen with either La3+ or 2APB alone. A representative of three experiments is shown. C, thrombin was added at 10 s to platelets in Ca2+-free buffer, and at 60 s, 1.0 mM Ca2+ was added to initiate Ca2+ influx. After 80 s, either 2APB, EGTA, or 2APB plus EGTA was added. A representative of four experiments is shown.

Time Course of the 2APB to Inhibit Thrombin- and Thapsigargin-Stimulated Ca²⁺ Influx and Basal Sr²⁺ Influx. The experiments presented above predominantly involved incubating platelets with 2APB for short periods (10-30 s) or adding 2APB together with either Ca²⁺ or Sr²⁺ at the same time (Figs. 7 and 9, A-C). The following experiments were performed to investigate just how rapid the effect of 2APB was in inhibiting the actions of thrombin and thapsigargin. The data in Fig. 10A show that 2APB caused an immediate and large decrease in the thrombin-mediated increase in [Ca²⁺]_i. The 2APB was added when the [Ca²⁺]_i had increased to its maximum level. For comparison purposes, 100 µM La³⁺ was also added to thrombin-stimulated platelets, and it also produced an immediate cessation in the elevation of [Ca²⁺]_i. The comparison of the effects of La³⁺ was used recently to show that LY294002 and farnesylcysteine analogs were not direct Ca²⁺-channel blockers in platelets (Rosado and Sage, 2000b,c). LY294002 had no effect on [Ca²⁺]_i, although farnesylcysteine analogs inhibited [Ca²⁺]_i only after a delay of 30 s, whereas La³⁺ produced an immediate inhibitory effect. La³⁺, a nonselective Ca²⁺-channel blocker, is unlikely to enter the platelet because of its positive charge; therefore, its action would be confined to plasma membrane Ca²⁺ channels and not intracellular Ca²⁺ channels (Hoth and Penner, 1993). The combination of La³⁺ plus 2APB did not produce any greater decrease in Ca²⁺ influx than that observed with either La³⁺ or 2APB alone; all these treatments produced identical decreases in [Ca²⁺]_i (Fig. 10A). These results imply that 2APB and La³⁺ were blocking the same channel(s) in the plasma membrane.

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Time course for the effects of La3+ and 2APB to inhibit Sr2+ influx in unstimulated platelets (no agonist). The influx of Sr2+ was initiated by adding 10 mM SrCl2 at 10 s. When Sr2+ influx was at a maximum rate (110 s), either La3+, 2APB, or La3+ plus 2APB was added. These treatments caused an immediate cessation in Sr2+ influx, consistent with La3+ and 2APB both blocking Ca2+ channels in the plasma membrane. A representative of four experiments is shown.

Lack of Effect of 2APB on the Ability of Ionomycin to Increase [Ca²⁺]_i. The action of the Ca²⁺ ionophore ionomycin to increase [Ca²⁺]_i in platelets should not be influenced by 2APB if 2APB were blocking Ca²⁺ channels in either the plasma membrane or ER, because ionomycin merely moves Ca²⁺ ions across membranes independent of Ca²⁺ channels. The data in Fig. 12 support this hypothesis, because the capacity of 2APB to influence the increase in [Ca²⁺]_i induced by ionomycin was minimally affected. Two methods were used to study the ability of ionomycin to increase [Ca²⁺]_i. In one approach, ionomycin was added to platelets in the presence of extracellular Ca²⁺; in the other approach, ionomycin was added to platelets in the absence of extracellular Ca²⁺ so that the ionomycin could mobilize intracellular Ca²⁺; then Ca²⁺ was added back to the platelets. The Ca²⁺ influx observed at this stage most probably represented the ability of ionomycin to translocate Ca²⁺ across the plasma membrane. The rate of increase in [Ca²⁺]_i was slightly inhibited, but the maximum effect on [Ca²⁺]_i was not affected using either protocol. This result also shows that 2APB does not influence the Ca²⁺/fura-2 fluorescence signal in platelets and therefore cannot account for the inhibitory effects of 2APB on [Ca²⁺]_i observed in this study.

View larger version (29K): [in this window] [in a new window]   Fig. 12.   The effect of 2APB on the ability of ionomycin to increase [Ca2+]i. Ionomycin (1.0 µM) was added to platelets in the presence of 1.0 mM extracellular Ca2+ in the presence or absence of 100 µM 2APB (added 10 s before data collection was started). 2APB reduced the rate of increase in [Ca2+]i but had no effect on the maximum effect. In another experiment, 1.0 µM ionomycin was added to platelets in the absence of extracellular Ca2+ in the presence or absence of 100 µM 2APB. After 200 s, Ca2+ was added to the platelets, and the increase in [Ca2+]i was measured. 2APB reduced the rate of increase in [Ca2+]i, but it had no effect on the maximum effect. A representative of three experiments is shown.

Pharmacophore Responsible for the Inhibition of Ca²⁺ Influx. We examined several compounds that are structurally related to 2APB for Ca²⁺ influx-blocking activity (Fig. 1). The first was diphenylboronic anhydride (DPBA), in which the two diphenylboronic groups are separated by an oxygen atom. The data in Fig. 13 show a dose response of DPBA to inhibit the ability of thrombin to increase [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺. The IC₅₀ value for DPBA to inhibit thrombin-mediated elevation of [Ca²⁺]_i when extracellular Ca²⁺ was either present or absent was approximately 2 µM. A greater inhibition (90% at 100 µM) of the increase in [Ca²⁺]_i was observed when extracellular Ca²⁺ was present compared with when Ca²⁺ was absent (60% at 100 µM). The IC₅₀ value for DPBA to inhibit [Ca²⁺]_i was approximately five times lower than the IC₅₀ value for 2APB to inhibit the thrombin-induced [Ca²⁺]_i increase in the presence and absence of Ca²⁺, which was 10 µM (Fig. 3). Therefore, it seems that the diphenylboronic moiety is the sole requirement for producing an inhibition of Ca²⁺ influx.

View larger version (27K): [in this window] [in a new window]   Fig. 13.   Dose response of DPBA and DPTTF to inhibit thrombin-mediated elevation in [Ca2+]i in the presence and absence of extracellular Ca2+. The dose-response experiments were performed in a manner similar to that described for 2APB in the legends to Figs. 2 and 3. The values are means ± S.E.M. from four to five separate experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 14.   Minimized structures of xestospongin C (top) and 2APB dimer (bottom) obtained by MM2 force field. Hydrogen atoms have been omitted for clarity.

Concluding Comments. This study demonstrates a lack of specificity of 2APB as a unique inhibitor of IP₃-induced Ca²⁺ release in human platelets. We predict that 2APB would have similar effects on other cells because many other cell types have been shown to possess both SOCC- and IP₃-induced Ca²⁺-release mechanisms. If 2APB had two sites of action, it would seem from this study to be an unusual Ca²⁺-channel antagonist, because it reduces agonist-stimulated increases in [Ca²⁺]_i by inhibiting both internal release of Ca²⁺ (Fig. 2B) and Ca²⁺ influx across the plasma membrane via SOCC.