©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ca-ATPase Isoforms of Platelets Are Located in Distinct Functional Ca Pools and Are Uncoupled by a Mechanism Different from That of Skeletal Muscle Ca-ATPase (*)

(Received for publication, May 8, 1995; and in revised form, July 5, 1995)

Simone Engelender Herman Wolosker Leopoldo de Meis (§)

From the Instituto de Ciencias Biomedicas, Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Ilha do Fundao, RJ 21941-590, Brasil

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Vesicles derived from the dense tubular system of platelets possess a Ca-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca regardless of the substrate used.

When acetyl phosphate was used in platelet vesicles, the transport of Ca was inhibited by Na, Li, and K; in sarcoplasmic reticulum vesicles, only Na caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles.

The catalytic cycle of the Ca-ATPase is reversed when a Ca gradient is formed across the vesicle membrane. The stoichiometry between active Ca efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles.

The coupling between ATP synthesis and Ca efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 µM), micromolar Ca concentrations, P(i) (5-20 mM), and MgATP (10-100 µM). Trifluoperazine also uncoupled the platelet Ca pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca, Mg, or K antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles.

The data indicate that the Ca-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.


INTRODUCTION

The Ca-ATPase found in the endoplasmic reticulum plays an important role in the maintenance of a low cytosolic Ca concentration in different cells. At least three genes encode the sarco/endoplasmic reticulum Ca-ATPase (SERCA). (^1)SERCA(1) Ca-ATPase isoform is expressed in fast skeletal muscle (1) . SERCA(2) gives rise to SERCA and SERCA isoforms by alternative splicing. SERCA is expressed in cardiac and slow skeletal muscle (2) , while SERCA is expressed in smooth muscle and represents a generic ``endoplasmic reticulum'' form that, together with SERCA(3), is found in several non-muscle cells(3, 4) .

The Ca-ATPase (SERCA(1)) found in vesicles derived from the sarcoplasmic reticulum has been studied extensively. This enzyme catalyzes the formation of a Ca gradient by translocating Ca from the medium into the vesicle lumen using energy derived from the hydrolysis of ATP(5) . The entire catalytic cycle of the ATPase can be reversed, so that the enzyme synthesizes ATP from ADP and P(i) using the energy derived from the Ca gradient. The synthesis of ATP is coupled with the release of Ca from the vesicles into the medium (5, 6, 7, 8, 9) . The reversal of the Ca pump is uncoupled by different drugs. This was first observed with arsenate, a phosphate analog that interacts with the catalytic site of the Ca ATPase, increasing the rate of Ca efflux and inhibiting the synthesis of ATP(10, 11, 12) . Similar to arsenate but in concentrations two orders of magnitude lower, a wide variety of hydrophobic drugs such as phenothiazines(13, 14, 15, 16) , local anesthetics (17) , and fatty acids (18) can also uncouple the Ca pump, greatly increasing the efflux of Ca from the vesicles. Ligands and substrates of the ATPase block the Ca efflux through the Ca pump promoted both by arsenate and hydrophobic drugs(11, 13, 14, 16) .

The dense tubular system found in blood platelets is a membranous network that retains a Ca transport ATPase. Like the enzyme found in muscle, it can catalyze both the hydrolysis and the synthesis of ATP(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) . Recently it has been shown that platelets express both SERCA and SERCA(3) Ca-ATPase isoforms(30, 31, 32) . It is not clear why different Ca-ATPase isoforms coexist in platelets and the possibility is then raised that the two isoforms may be located in functionally distinct Ca storage pools of the cell.

In this report we compare the kinetic properties of the sarcoplasmic reticulum Ca-ATPase (SERCA(1)) with those of platelets (SERCA and SERCA(3)). It is shown that arsenate and trifluoperazine uncouple the Ca pump of these two membrane systems in different manners. In platelets, but not in muscle, the substrate specificity and the sensitivity to arsenate indicate the possible existence of two different Ca storage pools.


MATERIALS AND METHODS

Outdated platelets were obtained from a blood bank in a concentrated form. Platelet vesicles derived from the dense tubular system were prepared as described by Le Peuch et al.(28) . Light sarcoplasmic reticulum vesicles derived from the longitudinal tubules of rabbit skeletal muscle were prepared according to Eletr and Inesi(33) . This vesicles preparation does not contain significant amounts of ryanodine/caffeine-sensitive Ca channels, nor does it exhibits the phenomenon of ``Ca-induced Ca release'' found in the heavy fraction of sarcoplasmic reticulum(14, 34) . The vesicles were stored in liquid nitrogen until use.

Protein concentration was estimated by the procedure of Lowry et al.(35) using bovine serum albumin as standard.

The Ca uptake was measured in medium containing 50 mM MOPS-Tris, pH 7.0, 100 mM KCl, 0.01-0.05 mg of protein/ml, and various concentrations of CaCl(2), MgCl(2), P(i), and ATP or acetyl phosphate as specified in the figure legends. The reaction was arrested by filtration through Millipore filters (0.45 µm)(36) . The protein retained on the filter was washed four times with 5 ml of 3 mM La(NO(3))(3), and the remaining radioactivity was counted in a scintillation counter.

For the Ca efflux experiments, the vesicles were preloaded with Ca in a medium containing 50 mM MOPS-Tris, pH 7.0, 5 mM MgCl(2), 20 mM P(i), 0.04-0.3 mM CaCl(2), 3 mM ATP or 10 mM acetyl phosphate, and 0.1 mg of protein/ml. After 30-45 min of incubation at 35 °C, the vesicles were centrifuged at 40,000 g for 40 min, the supernatant was discarded, and the walls of the tubes were blotted to minimize contamination by the residual Ca loading medium. The pellets were immediately resuspended in ice-cold water with four strokes of a glass homogenizer and diluted to a final concentration of 0.05-0.1 mg of protein/ml into a medium containing 50 mM MOPS-Tris, pH 7.0, and additions specified in the figure legends.

For the synthesis of ATP from ADP and P(i), the vesicles were loaded with nonradioactive Ca as described above and diluted in a medium containing 50 mM MOPS-Tris, pH 7.0, 5 mM EGTA, 5 mM MgCl(2), 10 units/ml hexokinase, 10 mM glucose, 5 mMP(i), and 0.2 mM ADP. The reaction was arrested with trichloroacetic acid to a final concentration of 10% (w/v). After centrifugation, an aliquot of the supernatant was withdrawn and excess P(i) was removed from the assay medium as phosphomolybdate complex with isobutyl alcohol/benzene (1:1, v/v). After six reextractions with acetone and isobutyl alcohol/benzene, an aliquot of the aqueous phase was counted in a scintillation counter(37) . The addition of hexokinase and glucose in the assay medium ensured that the ATP synthesized was converted to glucose 6-phosphate and maintained constant the ADP concentration in the medium through the experiment.

ATP hydrolysis was determined by measuring the release of P from [-P]ATP. The reaction was quenched with 2 volumes of a suspension of activated charcoal in 0.1 M HCl(37) . After centrifugation, aliquots of the supernatant containing P(i) were counted in scintillation counter. Mg-dependent ATPase activity was measured in the presence of 1 mM EGTA. The Ca-stimulated ATPase activity was determined by subtracting the Mg-dependent ATPase activity from the total ATPase activity measured in the presence of both Mg and Ca. The free Ca concentration was calculated as described by Fabiato and Fabiato (38) using the apparent association constants for Ca-EGTA determined by Schwartzenbach et al.(39) .

Heparin from porcine intestinal mucosa, D-myo-inositol 1,4,5,-trisphosphate and trifluoperazine were obtained from Sigma. Thapsigargin (LC Service, Woburn, MA) was dissolved in dimethyl sulfoxide. After dilution, the final concentration of dimethyl sulfoxide in the assay medium was less than 1%. Ca was purchased from DuPont NEN.


RESULTS

Ca Uptake

The Ca-ATPase found in sarcoplasmic reticulum vesicles can use different triphosphates, nucleosides, and acetyl phosphate as substrate(5) . The rate of Ca accumulation by sarcoplasmic reticulum vesicles varied depending on whether ATP, ITP, or acetyl phosphate was used, but when the steady-state was reached, the maximum amount of Ca accumulated by the vesicles was the same regardless of the substrate (Fig. 1B). We found that vesicles prepared from platelets can also use acetyl phosphate and ITP as substrate, but different from the muscle Ca-ATPase, the maximum level of Ca accumulated with these substrates was 2-3-fold smaller than that measured with ATP (Fig. 1A).


Figure 1: Substrate specificity of Ca-ATPase from platelet vesicles (A) and sarcoplasmic reticulum vesicles (B). The Ca uptake was measured at 35 °C in a medium containing 50 mM MOPS-Tris, pH 7.0, 10 mM MgCl(2), 10 mM P(i), 40 µMCaCl(2), 100 mM KCl, 0.05 (A) or 0.01 (B) mg of protein/ml and either 3 mM ATP (), 10 mM acetyl phosphate (), or 5 mM ITP (bullet). The values are representative of three different experiments performed with three different vesicle preparations.



With the use of acetyl phosphate, but not with ATP, a difference in the sensitivity to monovalent cations was observed. With ATP, monovalent cations had practically no effect in the Ca accumulation measured with either platelet or sarcoplasmic reticulum vesicles (Table 1). However, the Ca uptake supported by acetyl phosphate was inhibited to different extents depending on the vesicle preparation used. In platelet vesicles, inhibition was observed with all three cations, so that: Na = Li > K in order of inhibitory activity (Table 1). In sarcoplasmic reticulum vesicles, only Na significantly inhibited Ca uptake.



Thapsigargin is a highly specific inhibitor of SERCA isoforms and has no effect on plasma membrane Ca ATPase(40, 41, 42) . Nanomolar concentrations of thapsigargin abolished the Ca uptake of platelet (Fig. 2) and sarcoplasmic reticulum vesicles (not shown) loaded with the use of either ATP or acetyl phosphate. This indicates that the Ca accumulation measured in Fig. 1and Table 1was mediated by SERCA isoforms in the dense tubules of platelets and not by plasma membrane contaminants.


Figure 2: Inhibition of Ca uptake by thapsigargin in platelet vesicles. The assay medium contained 50 mM MOPS-Tris, pH 7.0, 5 mM MgCl(2), 20 mM P(i), 60 µMCaCl(2), 100 mM KCl, 0.05 mg of protein/ml, and either 4 mM ATP (bullet) or 10 mM acetyl phosphate (). The reaction time was 45 min at 35 °C. The values are the means ± S.E. of three different experiments using three different vesicle preparations. 100% Ca uptake was 170 ± 15 with ATP and 50 ± 5 with acetyl phosphate.



Passive Ca Efflux

A slow release of Ca is observed when vesicles previously loaded with Ca are diluted in medium containing EGTA and none of the ligands of the ATPase(5) . In earlier reports (9, 13, 14) it has been shown that this passive Ca efflux from sarcoplasmic reticulum vesicles is decreased when either Ca, Mg, K, or thapsigargin is added to the efflux medium (Table 2). As previously discussed(9, 13, 14) , this indicates that, in sarcoplasmic reticulum vesicles, passive Ca efflux occurs through the Ca-ATPase. The passive Ca efflux from platelet vesicles was much slower and was not inhibited by Ca, Mg, or K (Table 2). Thapsigargin inhibited the efflux from platelet vesicles but its effect was less pronounced than that measured in sarcoplasmic reticulum (Table 2). The effects observed in Table 2were the same regardless of whether the vesicle preparations were loaded using ATP or acetyl phosphate as substrate (data not shown).



Reversal of the Ca Pump

The rate of Ca efflux is enhanced when the loaded vesicles are incubated in a medium that contains ADP, P(i), and Mg in addition to EGTA. The difference between the efflux in this case and the passive efflux with only EGTA present is termed active Ca efflux and is coupled to the synthesis of ATP (5) . In sarcoplasmic reticulum vesicles, the stoichiometry between Ca release and ATP synthesis is two (5, 8) (Fig. 3B). The catalytic cycle of platelet Ca ATPase could also be reversed(29) , but different from sarcoplasmic reticulum, the stoichiometry found between active Ca efflux and ATP synthesis was one (Fig. 3A), regardless of the substrate used to load the vesicles. For platelet vesicles loaded with acetyl phosphate, the stoichiometry found was 1.20 ± 0.10 (&Xcjs1171; ± S.E., n = 4) (Fig. 3A), and for vesicles loaded with ATP it was 1.19 ± 0.08 (n = 5).


Figure 3: Active Ca efflux (bullet) and ATP synthesis () in platelet and sarcoplasmic reticulum vesicles. Platelet vesicles (A) or sarcoplasmic reticulum vesicles (B) loaded with Ca using acetyl phosphate were diluted in medium containing 50 mM MOPS-Tris, pH 7.0, 5 mM EGTA, 5 mM MgCl(2), 5 mM P(i), 20 µg/ml hexokinase, 10 mM glucose, and 0.1 mg of protein/ml. Active Ca efflux was calculated by subtracting the efflux measured in the absence of ADP from that measured in the presence of ADP. The values are the means ± S.E. of four different experiments with three different preparations.



Uncoupling of the Ca Pump by Arsenate

Arsenate uncouples the reversal of the sarcoplasmic reticulum pump. It competes with P(i) for the catalytic site of the SERCA(1) ATPase and enhances the rate of passive Ca efflux at the same time that it inhibits the synthesis of ATP(10, 11, 12) . This is observed regardless of whether the vesicles are loaded with ATP or acetyl phosphate(12) . Unlike vesicles derived from muscle, platelet vesicles respond to arsenate with a pronounced increase in Ca efflux only after loading with acetyl phosphate (Figs. 4 and 5). Note that, in these experiments, the substrate used in the loading mixture was removed by centrifugation before the addition of preloaded vesicles to the efflux medium. These data and those of Fig. 1suggest that platelet vesicles are composed of two distinct populations in which only the fraction of platelet vesicles that are loaded with acetyl phosphate (Fig. 1A) can be uncoupled by arsenate (Fig. 4A). The difference in the effect of arsenate on platelet vesicles loaded with acetyl phosphate and ATP is probably not related to the amount of Ca retained by the vesicles. The rate of Ca efflux depends on the concentration of free Ca inside the vesicles. Inorganic phosphate was used as a calcium-precipitating agent when the vesicles were loaded. Thus, the total calcium accumulated refers to the calcium phosphate retained by the vesicles, but the free Ca concentration will always be determined by the solubility product of calcium phosphate and should be the same regardless of the amount of calcium phosphate inside the vesicles(34) . Accordingly, the slower Ca efflux of platelet vesicles when compared to sarcoplasmic reticulum vesicles (Table 2) is probably not related to different amount of Ca in the vesicles, but to a difference of the number of ATPase units available in the membrane of the two vesicles preparations. In fact, the Ca-ATPase of platelet vesicles accounts for 2-4% of the total membrane protein(43) , while in sarcoplasmic reticulum vesicles it represents 70-80% of the total protein(5) .


Figure 4: Arsenate-induced Ca efflux in platelet vesicles; Blockage by thapsigargin. Platelet vesicles loaded using acetyl phosphate (A) or ATP (B) were diluted in a medium containing 50 mM MOPS-Tris, pH 7.0, 5 mM EGTA, 5 mM MgCl(2), 0.05 mg of protein/ml either without additions (), with 10 mM arsenate (bullet), or 2 µM thapsigargin plus 10 mM arsenate (). The figure shows the Ca remaining in the vesicles after different incubation intervals. The values are the means of 18 different experiments. The S.E.s were less than 4%. Error bars are smaller than the symbols.



The effect of arsenate on vesicles loaded with acetyl phosphate was completely antagonized by 2 µM thapsigargin (Fig. 4). This indicates that the enhancement of Ca efflux by arsenate is mediated by the platelet Ca-ATPase.

In sarcoplasmic reticulum, the concentration of arsenate required to promote half-maximum stimulation of Ca efflux was 4.2 mM, regardless of the substrate used to load the vesicles (Fig. 5B). Similarly, in platelet vesicles loaded with acetyl phosphate, half-maximum stimulation was obtained with 3.6 mM arsenate (Fig. 5A).


Figure 5: Effect of arsenate on Ca efflux from platelet and sarcoplasmic reticulum vesicles. Platelet vesicles (A) and sarcoplasmic reticulum vesicles (B) loaded using acetyl phosphate () or ATP (bullet) were diluted in a medium identical to Fig. 4in the presence of increasing arsenate concentrations. The figure shows the percent of Ca remaining in the vesicles after 15 min (A) or 5 min (B). The values are representative of three different experiments performed with three different vesicle preparations.



In sarcoplasmic reticulum vesicles, the effect of arsenate requires magnesium and is antagonized by micromolar Ca concentrations, P(i), and MgATP(10, 11) . The same was observed for the fraction of platelet vesicles loaded by acetyl phosphate (Fig. 6-8), with the only difference being that the concentration of MgATP required to antagonize the effect of arsenate in platelet vesicles was one order of magnitude smaller than that required for sarcoplasmic reticulum vesicles (compare Fig. 8, A and B).


Figure 6: Concentration dependence of MgCl(2) (A) and CaCl(2) (B) effects on arsenate-induced Ca efflux from platelet vesicles. Platelet vesicles were loaded using acetyl phosphate and diluted in a medium containing 50 mM MOPS-Tris, pH 7.0, 5 mM EGTA, 0.05 mg 0f protein/ml, either in the absence () or in the presence (bullet) of 10 mM arsenate. The reaction time was 15 min (A) or 20 min (B). The values are representative of three different experiments performed with three different vesicle preparations.




Figure 8: Effect of MgATP on Ca efflux from platelet vesicles (A) and sarcoplasmic reticulum vesicles (B). Vesicles were loaded using acetyl phosphate and diluted in a medium identical to that in Fig. 4, in the presence of increasing MgATP concentrations. The figure shows the Ca remaining in the vesicles after 15 (A) or 5 min (B). The values are representative of three different experiments performed with three different vesicle preparations.



For platelet vesicles loaded with ATP, arsenate did not activate the efflux of Ca even after the addition of hexokinase (50 units/ml) and glucose (20 mM) to the efflux medium to drain residual ATP.

Effect of Trifluoperazine

Hydrophobic drugs such as trifluoperazine are more effective than arsenate in uncoupling the sarcoplasmic reticulum Ca-ATPase. In addition to increasing passive Ca efflux, trifluoperazine inhibits Ca uptake and ATPase activity in sarcoplasmic reticulum vesicles(13, 14, 15, 16, 44) . These effects were also observed with platelet vesicles, where trifluoperazine inhibited both Ca uptake and ATPase activity in lower concentrations than those necessary for sarcoplasmic reticulum vesicles (Fig. 9). Half-maximal inhibition of Ca uptake (Fig. 9A) was attained with one-fifth the concentration of trifluoperazine required for inhibition of ATPase activity (Fig. 9B). White and Raynor (45) reported that trifluoperazine inhibits Ca uptake in platelet vesicles and proposed that this effect was related solely to inhibition of Ca influx, since it did not affect Ca efflux. In our experimental conditions, inhibition of Ca uptake is probably related to enhancement of Ca efflux ( Fig. 10and 11). Like arsenate, trifluoperazine inhibited the synthesis of ATP and active Ca efflux from platelet vesicles (data not shown). A puzzling finding was that, different from arsenate, trifluoperazine uncoupled the Ca pump of platelet vesicles regardless of whether the vesicles were loaded using ATP or acetyl phosphate (data not shown).


Figure 9: Inhibition of Ca uptake and Ca-ATPase activity by trifluoperazine in platelet and sarcoplasmic reticulum vesicles. A, Ca uptake was measured at 35 °C in medium containing 50 mM MOPS-Tris, pH 7.0, 2 mM MgCl(2), 2 mM ATP, 60 µMCaCl(2), 10 mM P(i), increasing concentrations of trifluoperazine and either 0.1 mg/ml platelet vesicles () or 0.01 mg/ml sarcoplasmic reticulum vesicles (bullet). The reaction time was 40 min for platelet vesicles and 5 min for sarcoplasmic reticulum vesicles. The values represent the percent of Ca uptake without trifluoperazine: 180 nmol of Ca/mg for platelet vesicles and 2.8 µmol of Ca/mg for sarcoplasmic reticulum vesicles. B, ATPase activity was measured in medium containing [-P]ATP instead of radioactive Ca. The values represent the percent of ATPase activity without trifluoperazine: 540 nmol of P(i)/mg for platelet vesicles and 1.4 µmol of P(i)/mg for sarcoplasmic reticulum vesicles. The values are representative of three different experiments performed with three different vesicle preparations.




Figure 10: Effect of thapsigargin and cations on trifluoperazine-induced Ca efflux from platelet vesicles (A and C) and sarcoplasmic reticulum vesicles (B and D). Vesicles were preloaded with CaCl(2) using ATP and diluted to a final concentration of 50 µg/ml in medium containing 50 mM MOPS-Tris, pH 7.0, and 5 mM EGTA. A and B, no addition (), 40 µM trifluoperazine (bullet), or 40 µM trifluoperazine plus 1 µM thapsigargin (). C and D, no addition (), 50 µM trifluoperazine (bullet), 50 µM trifluoperazine plus 0.1 mM CaCl(2) instead of EGTA (), 50 µM trifluoperazine plus 10 mM MgCl(2) (), or 50 µM trifluoperazine plus 100 mM KCl (). The figure shows the Ca remaining in the vesicles after different incubation intervals. The values are representative of three different experiments performed with three different vesicle preparations.



In contrast to its effects in sarcoplasmic reticulum, the effect of trifluoperazine in platelet vesicles was not antagonized by thapsigargin or cations ( Fig. 10and Fig. 11). Even the presence of Ca and K together did not block the effect of trifluoperazine on Ca efflux from platelet vesicles (Fig. 11A).


Figure 11: Concentration dependence of trifluoperazine-induced Ca efflux from platelet and sarcoplasmic reticulum vesicles; Effect of Ca and K. Platelet vesicles (A) and sarcoplasmic reticulum vesicles (B) were loaded using ATP and diluted in a medium identical to that used in Fig. 4in the presence of increasing trifluoperazine concentrations, either with no other additions (), 0.1 mM CaCl(2) instead of EGTA (bullet), 100 mM KCl (), or 0.1 mM CaCl(2) instead of EGTA plus 100 mM KCl (up triangle, filled). The figure shows the Ca remaining in the vesicles after 5 min (A) or 1 min (B). The values are representative of three different experiments performed with three different vesicle preparations.



IP(3) and Heparin

IP(3) (5 µM) promoted a 7-9% increase in Ca efflux from platelet vesicles (data not shown). The increase in rate in our experiments is similar to that reported in the literature (24) and is severalfold smaller than that caused by either arsenate or trifluoperazine. The effect of IP(3) was the same in vesicles loaded using ATP or acetyl phosphate. Heparin blocks the binding of IP(3) to its receptor and abolishes Ca release through the IP(3)-sensitive Ca channel(46, 47) . However, addition of heparin (up to 100 µg/ml) did not inhibit the action of either arsenate or trifluoperazine (data not shown).


DISCUSSION

The present study shows kinetic differences between the Ca-ATPase isoforms of skeletal muscle and platelets. Modulation of Ca uptake and Ca efflux by cations ( Table 1and Table 2), inhibition of passive Ca efflux by thapsigargin (Table 2) and the Ca/ATP stoichiometry measured during reversal of the pump (Fig. 3) distinguished the sarcoplasmic reticulum Ca-ATPase isoform from that found in platelet vesicles.

The uncoupling effect of arsenate differentiated platelet vesicles loaded using ATP from those loaded using acetyl phosphate. Arsenate increased passive Ca efflux only from platelet vesicles loaded with acetyl phosphate (Fig. 4), and this efflux was antagonized by thapsigargin (Fig. 4), micromolar Ca concentrations (Fig. 6B), P(i) (Fig. 7), and MgATP (Fig. 8). These data indicate that vesicles loaded with acetyl phosphate contain a Ca-ATPase isoform with properties different from those found in vesicles loaded with ATP. Other cells, such as bovine chromaffin cells (48) possess two Ca stores with distinct Ca-ATPases in each of them.


Figure 7: Reverse of arsenate effect by P(i). Platelet vesicles loaded using acetyl phosphate were diluted in a medium identical to that in Fig. 3in the presence of increasing P(i) concentrations, either in the absence (, ) or in the presence (bullet, up triangle, filled) of 10 mM arsenate. A, active Ca efflux; B, ATP synthesis. The reaction time was 20 min. The values are representative of three different experiments performed with three different vesicle preparations.



Trifluoperazine also uncoupled the Ca pump of platelet vesicles, but unlike arsenate, it did not distinguish vesicles loaded with ATP from those loaded with acetyl phosphate. However, this drug did differentiate between muscle and platelet Ca-ATPase isoforms, since thapsigargin and cations did not antagonize the effect of trifluoperazine in platelet vesicles ( Fig. 10and Fig. 11). The trifluoperazine concentrations used are at least 150-200-fold in excess in molar basis to the Ca-ATPase content of either muscle or platelet vesicles preparations. Thus, the differences in sensitivity to trifluoperazine of the two vesicles preparations are not related to differences in Ca-ATPase content.

Uncoupling of the platelet Ca-ATPase by arsenate and trifluoperazine produced a greater increase in Ca efflux than that promoted by IP(3). This suggests that the number of Ca-ATPase units found in the vesicle membranes is greater than the number of IP(3) receptors.

The effects of arsenate and trifluoperazine indicate that Ca can be released from platelet vesicles through the Ca ATPase. This pathway might serve to mobilize Ca in the cell. It is noteworthy that arsenate and trifluoperazine exhibit distinct effects depending on the tissue studied, even allowing identification of different functional compartments in platelets. The physiological implications of different Ca pools in platelets and other tissues, such as bovine chromaffin cells, are not clear at present.


FOOTNOTES

*
This work was supported by grants from Programa de Auxilio ao Desenvolvimento de Ciêancia e Tecnologia, Conselho Nacional de Desenvolvimento Cient&ıacute;fico e Tecnológico and Financiadora de Estudos e Projetos. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 5521-270-8647.

(^1)
The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca-ATPase; IP(3), inositol 1,4,5-trisphosphate; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Sorenson for helpful discussion during the preparation of the manuscript and to Antonio Carlos M. da Silva and Valdecir Antunes Suzano for technical assistance. We are indebted to the staff of the Blood Bank at Hospital Universitário Clementino Fraga Filho for providing the platelet concentrates.


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