(Received for publication, May 8, 1995; and in revised form, July 5, 1995)
From the
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
(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.
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). (
)SERCA
Ca
-ATPase isoform is expressed in fast skeletal
muscle (1) . SERCA
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
, is found in several non-muscle
cells(3, 4) .
The Ca-ATPase
(SERCA
) 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
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
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
) with those of
platelets (SERCA
and SERCA
). 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.
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
, MgCl
, P
, 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
)
,
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
, 20 mM P
, 0.04-0.3 mM CaCl
, 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
, 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
, 10 units/ml hexokinase, 10 mM glucose, 5 mM
P
, 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
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
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.
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
, 10 mM
P
, 40 µM
CaCl
, 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 (
). 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
, 20
mM P
, 60 µM
CaCl
, 100 mM KCl, 0.05 mg of
protein/ml, and either 4 mM ATP (
) 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.
Figure 3:
Active Ca efflux
(
) 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
, 5 mM P
, 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.
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
, 0.05 mg of protein/ml
either without additions (
), with 10 mM arsenate
(
), 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 (
) 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
, 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 (A) and CaCl
(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
(
) 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.
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 mM
ATP, 60 µM
CaCl
, 10 mM P
, increasing concentrations of trifluoperazine and
either 0.1 mg/ml platelet vesicles (
) or 0.01 mg/ml sarcoplasmic
reticulum vesicles (
). 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
/mg for
platelet vesicles and 1.4 µmol of P
/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
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
(
), or 40 µM trifluoperazine plus 1 µM thapsigargin (
). C and D, no addition
(
), 50 µM trifluoperazine (
), 50 µM trifluoperazine plus 0.1 mM CaCl
instead of
EGTA (
), 50 µM trifluoperazine plus 10 mM MgCl
(
), 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
instead of EGTA
(
), 100 mM KCl (
), or 0.1 mM CaCl
instead of EGTA plus 100 mM KCl (
). 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.
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
(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. Platelet vesicles loaded using acetyl phosphate were
diluted in a medium identical to that in Fig. 3in the presence
of increasing P
concentrations, either in the absence
(
,
) or in the presence (
,
) 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
. This suggests that
the number of Ca
-ATPase units found in the vesicle
membranes is greater than the number of IP
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.