(Received for publication, July 31, 1995; and in revised form, December 18, 1995)
From the
Prostaglandin I (PGI
) and sodium
nitroprusside (SNP) induce a rapid decay of the thrombin-promoted
increase of [Ca
]
in
aspirin-treated platelets incubated in the absence of external
Ca
. The mechanism of their effect was studied with a
new method which utilizes ionomycin to increase
[Ca
]
, followed by
bovine serum albumin (BSA) to remove the Ca
ionophore. The rapid decay of
[Ca
]
after BSA is
mostly due to the reuptake into the stores, since it is strongly
inhibited by the endomembrane Ca
-ATPase inhibitor
thapsigargin. PGI
and SNP are without effect on the
BSA-promoted decay both with and without thapsigargin, showing that
they do not affect the activity of the Ca
-ATPases.
The fast decay of [Ca
]
after BSA is decreased by thrombin which produces the
Ca
releaser inositol 1,4,5-trisphosphate
(InsP
), thus counteracting the activity of the endomembrane
Ca
pump. When added after thrombin, PGI
and SNP accelerate the BSA-activated decay of
[Ca
]
. However, under
the same conditions, they do not decrease the concentration of
InsP
. In saponin-permeabilized platelets, cAMP and cGMP
counteract the Ca
release induced by exogenous
InsP
. Their inhibitory effect disappears at high InsP
concentrations. This demonstrates that PGI
and SNP
potentiate Ca
reuptake by inhibiting the InsP
receptor. Two bands of approximately 260 kDa are recognized by a
monoclonal antibody recognizing the C-terminal region of the InsP
receptor. Both are phosphorylated rapidly, the heavier more
intensely, in the presence of PGI
and SNP. The
phosphorylation of the InsP
receptor is fast enough to be
compatible with its involvement in the inhibition of the receptor by
cyclic nucleotides.
The activation of platelets is stimulated or inhibited by
numerous hormones, drugs, eicosanoids, and other vasoactive substances.
Agonists such as thrombin, thromboxane, vasopressin,
platelet-activating factor, and ADP elevate the cytosolic free
Ca and stimulate the activity of myosin light chain
kinase and PKC (
)resulting in platelet adhesion,
aggregation, and degranulation(1, 2) .
Most
platelet agonists activate phospholipase C and elevate
[Ca]
by an
InsP
-dependent release of Ca
from the
intracellular stores, as well as stimulation of the entry of
extracellular
Ca
(2, 3, 4, 5) .
The agonist-releasable stores appear to be of two different types,
distinguishable by the sensitivity of their
Ca
-ATPases to low thapsigargin (Tg) concentrations or
to 2,5-di-tert-butylhydroquinone (or high Tg concentrations),
respectively(6, 7, 8) .
The modes of
Ca entry are controversial. It is generally agreed
that a substantial influx is activated by a signal generated by the
depletion of the intracellular stores (capacitative Ca
influx) (9) also in the absence of agonist, e.g. by treatment with the endomembrane Ca
-ATPase
inhibitor Tg(10, 11) ; such a signal could be a small
molecule released together with the Ca
ions from the
stores(12, 13) , but also other possibilities cannot
be excluded (14, 15, 16) . Receptor-mediated
influx systems are also operative. ADP induces a very fast
Ca
entry which precedes the release from the
intracellular stores and the subsequent second phase of Ca
entry(4) . We recently found that the occupancy of the
thrombin receptor activates a store-independent Ca
influx (17) .
Ca efflux from
platelets is operated by a Ca
-ATPase, whose activity
is potentiated by the activation of PKC (18, 19) as
well as by the depletion of the stores (19) .
The cyclic
nucleotides cAMP and cGMP exert multiple inhibitory actions on platelet
activation. cAMP was shown to decrease the binding of thrombin to its
receptor on human platelets(20) . In the presence of cAMP, the
activation of phospholipase C (and hence the production of InsP and diacylglycerol) by the agonist is depressed, leading to the
inhibition of the increment of
[Ca
]
and of
PKC-dependent phosphorylations(21) . The action of cAMP is also
on events distal to the activation of phospholipase C since platelet
aggregation and secretion induced by the Ca
ionophore
ionomycin or by phorbol esters are also inhibited by the cyclic
nucleotide(22, 23, 24) . cAMP also increases
the incorporation of diacylglycerol into
phosphatidylinositol(25) .
Similar inhibitory actions were
reported for cGMP, which interferes with the agonist-induced activation
of phospholipase C (26, 27, 28, 29, 30) and
also affects events distal to the increase of
[Ca]
(31, 32) .
Furthermore, cGMP potentiates theaction of cAMP elevating agents, by
inhibiting the cAMP phosphodiesterase(33) .
The cyclic
nucleotides also interfere with the Ca influx. We
recently reported that cAMP and cGMP inhibit the thrombin-activated
Ca
influx in platelets, without interfering with the
Ca
influx secondary to the depletion of the
intracellular stores(17) . An inhibition by cAMP and cGMP has
been reported recently on Ca
influx activated by
thromboxane(34) . On the contrary, the ADP-activated
Ca
influx has been reported to be insensitive to
cAMP(3) .
The action of cAMP and cGMP on Ca movements to and from the intracellular deposits is controversial
and has received considerable attention. The activity of PKA was
reported to be facilitatory, or even necessary, for Ca
release by InsP
from isolated platelet membrane
vesicles(35) . This conclusion was refuted(36) . The
catalytic subunit of PKA was reported to stimulate Ca
uptake by platelet membrane
vesicles(35, 37, 38) . This effect was
correlated with the phosphorylation of a protein tentatively identified
with phospholamban, the known promoter of Ca
transport in cardiac muscle membranes, but that identity was
disputed(39) . On the other hand, dibutyryl cAMP, and various
prostaglandins and forskolin, are known to stimulate adenylate cyclase
and reverse the Ca
mobilization produced by platelet
agonists(40, 41, 42) . Furthermore, the
addition of cAMP to saponin-permeabilized platelets was reported to
decrease the amount of Ca
released by the
InsP
-sensitive stores (43) . Similar results were
obtained with the catalytic subunit of PKA (4) .
Finally, it
was also proposed that cAMP and cGMP potentiate the action of the
plasma membrane Ca-ATPase(45) .
The
present study was performed to settle some controversy concerning the
action of cAMP and cGMP on Ca movements between
cytosol and the stores. It is shown that both cyclic nucleotides are
without effect on the Ca
pumps (including the plasma
membrane Ca
pump) and that their action is to prevent
the Ca
-releasing activity of InsP
. Both
cAMP and cGMP promote the phosphorylation of the InsP
receptor.
Figure 7:
Western blot identification of the
InsP receptor (A) and its phosphorylation by
PGI
(C). A, recognition of the InsP
receptor with an anti-InsP
monoclonal antibody. B, Coomassie blue pattern of platelet proteins (lane
2); the monomeric form of hemocyanine from Octopus vulgaris (250 kDa) was used as high molecular mass standard protein in lane 1. C, autoradiographic pattern; platelets were incubated
with 0.25 unit/ml thrombin for 10 s (lane 1), followed by 0.7
µg/ml PGI
for 10 s (lane 2), 30 s (lane
3), 60 s (lane 4), 300 s (lane 5). GF 109 203X
(3 µM) was added 3 min before thrombin. The reported
pattern is typical of at least 5 different experiments obtained from
different platelet preparations.
Figure 8:
Time course of the 260-kDa
(InsP) and of the 47-kDa protein phosphorylation by
PGI
, SNP, and thrombin.
, thrombin (0.25 unit/ml)
added at zero time. GF 109 203X (3 µM) was added 3 min
before thrombin. &cjs2106;, thrombin added at zero time followed 10 s
later by PGI
. GF 109 203X was added 3 min before thrombin.
&cjs2098;, thrombin added at zero time followed 10 s later by SNP. GF
109 203X was added 3 min before thrombin.
, thrombin added at
zero time. The data are collected from at least 5 different experiments
similar to those reported in Fig. 4B. The baseline
counts/min prior to the addition of thrombin were subtracted from all
the points.
Figure 4:
Platelet InsP elevated by
thrombin is not affected by the subsequent addition of PGI
or SNP. Thrombin (
) was added at zero time followed at 10 s,
where indicated, by PGI
(
) or SNP (
). Data are
in triplicate from 2 different
preparations.
Figure 1:
PGI and SNP stimulate the
decay of [Ca
]
elevated
by thrombin. Indicated are the additions of thrombin (Thr,
0.25 unit/ml), thapsigargin (Tg, 200 nM), and
ionomycin (IONO, 50 nM). The dotted line indicates the progress of the Ca
trace upon
addition of PGI
(0.6 µg/ml) or SNP (60
µM). The traces are representative of duplicate
experiments with at least 5 different preparations with similar
results.
Figure 2:
PGI and SNP have no effect on
the endoplasmic reticulum and plasma membrane
Ca
-ATPases. They inhibit the thrombin-induced
increase of Ca
efflux from the stores. Ca
discharge from the intracellular stores was induced with
ionomycin (IONO, 400 nM). The latter was then removed
with BSA (BSA, 2 mg/ml). The traces following the addition of
PGI
or SNP are indicated by the dotted lines. The
temperature was 20 °C. The traces are representative of duplicate
experiments with at least 5 different preparations with similar
results.
The
addition of PGI or SNP prior to ionomycin has no effect on
the ionophore-induced increase of
[Ca
]
and importantly also has
no effect on the rate of decrease of
[Ca
]
upon addition of BSA, both
in the absence (Fig. 2A) and presence (Fig. 2B) of Tg. Accordingly, the refilling of the
stores after BSA (in the experiment without Tg, Fig. 2A), as measured by the increase of
[Ca
]
following the addition of
Tg, is unmodified by PGI
and SNP. This is more clearly
shown (with an expanded time scale) in Fig. 3, where, after
ionomycin, BSA was supplemented both at submaximal and at maximal
concentrations. At the lower BSA concentrations, some ionomycin is left
unbound and activates variable degrees of Ca
cycling.
In these conditions, the combined action of the Ca
pumps and the ionomycin-induced leaks leads to the establishment
of intermediate levels of Ca
accumulation in the
stores and to intermediate
[Ca
]
. The rate of decay of
[Ca
]
decreases and the steady
state [Ca
]
increases with
decreasing BSA. In no case does the inclusion of PGI
or SNP
modify the decay rate or the final steady state. A stimulation of the
pump would have resulted in an increased decay rate of
[Ca
]
and a decreased final
steady state. These experiments show that the cyclic nucleotides do not
interfere with the operation of the two endomembrane
Ca
-ATPases (6, 7, 8) nor of
the plasma membrane Ca
pump.
Figure 3:
PGI and SNP do not affect the
rate of Ca
uptake by endoplasmic reticulum
Ca
-ATPase. Ca
discharge from the
intracellular stores was induced with ionomycin (IONO, 500
nM). The latter was then partially or totally removed with
different concentrations of BSA leading to variable degrees of
Ca
cycling. The traces with PGI
or SNP
are indicated by the dotted lines. The temperature was 20
°C.
Since the cyclic nucleotides appear not to modify
the activity of the Ca pumps, but rather to oppose
the effect of InsP
, we measured the thrombin-induced
production of InsP
and the effect of supplementing
PGI
or SNP, after thrombin, on the platelet InsP
content. As shown in Fig. 4, InsP
increases
sharply soon after the addition of thrombin, to progressively decrease
to a lower steady state value. The addition of PGI
or SNP
10 s after thrombin, at a time when InsP
has reached its
maximum, is without effect on the InsP
levels which remain
undistinguishable from those observed with thrombin alone. This finding
shows that the cAMP and cGMP potentiation of Ca
uptake into the stores (Fig. 1) is mediated by a decrease
of Ca
efflux rather than by an increase in influx (Fig. 2), and this takes place with no variation of InsP
concentration.
To monitor directly the effect of cAMP and cGMP
on the Ca release promoted by exogenous
InsP
, we also performed experiments in
saponin-permeabilized platelets. The amount of saponin required for the
permeabilization was variable and had to be adjusted in each platelet
preparation. A large Ca
uptake into the endoplasmic
reticulum is promoted by ATP upon permeabilization. Once the loading of
the stores is completed, Ca
is released stepwise by
graded pulses of InsP
. The Ca
released by
low concentrations of InsP
is strongly decreased by cAMP.
The inhibition is frequently slightly potentiated further by the
inclusion of PKA (the effect, however, was not appreciated in all
preparations). This may be expected since saponin permeabilizes the
plasma membrane to large molecules, thus allowing the loss into the
medium of many proteins and cofactors. The effect of cAMP disappears at
high InsP
concentrations. A typical experiment is reported
in Fig. 5, and the cumulative results from several different
platelet preparations are reported in Fig. 6(the points are
taken from experiments performed in the presence and absence of
exogenous PKA). Similar experiments with cGMP gave essentially
superimposable results.
Figure 5:
Fluo 3 measurement of the
InsP-induced Ca
release from
permeabilized platelets: inhibition by cAMP. Platelets were treated as
described under ``Experimental Procedures.'' When present,
cAMP was 20 µM, and PKA 100
units/ml.
Figure 6:
cAMP inhibition of the
InsP-induced release of Ca
from
permeabilized platelets is inhibited by cAMP. The points were collected
from experiments as in Fig. 7from at least 13 different
platelet preparations. Each point represents the mean value expressed
as percent of total Ca
released ± S. D. (bars) of at least 9 different determinations: *,**, and ***
indicate that cAMP and control are significantly different with a p < 0.05, < 0.01, < 0.005, respectively, calculated by the
Student's t test. The points with cAMP are taken from
experiments conducted in the presence as well as in the absence of
added PKA, since the two conditions were not statistically
significantly different overall.
, control;
,
cAMP.
The experiments reported in this study were performed in
order to determine the relative potency and the regulation by the
cyclic nucleotides cAMP and cGMP of the systems involved in the decay
of platelet [Ca]
. The effect of
the cyclic nucleotides is interesting since increasing their
concentration after an agonist-dependent Ca
release
from the stores potently stimulates the disappearance of
[Ca
]
. The experiments were
performed in Ca
-free media to avoid interferences
from Ca
influx.
In order to avoid unwanted
interferences by agonists and inhibitors on Ca movements, we introduced a new method which utilizes ionomycin to
deplete the deposits and increase
[Ca
]
, followed by BSA to
terminate the action of the Ca
ionophore. Using this
technique, we reached the following conclusions.
1. Upon addition of
BSA, the decrease of [Ca]
previously elevated by ionomycin is very rapid, such that also at
20 °C it is completed in less than 20 s.
2. The activities of
the endomembrane and plasma membrane Ca-ATPases
responsible for the decay of [Ca
]
can be readily discriminated by including Tg that specifically
inhibits the endomembrane Ca
-ATPases. These
experiments show that pumping into the deposits prevails strongly over
pumping across the plasma membrane.
3. Including PGI or
SNP is without visible effect on the BSA-induced decay of
[Ca
]
both in the absence and
presence of Tg. This shows that cAMP and cGMP do not interfere with the
activity of either the endomembrane or the plasma membrane
Ca
-ATPases.
4. In the absence of Tg, the
BSA-activated decay of [Ca]
is
strongly decreased if thrombin is supplemented together with BSA. This
shows that the thrombin-activated production of InsP
, by
inducing a (BSA-insensitive) Ca
efflux from the
stores, counteracts the action of the endomembrane Ca
pumps, thus decreasing the [Ca
]
decrease rate. Adding PGI
or SNP along the
[Ca
]
decay trace (slowed down
by thrombin) promptly accelerates the decay of
[Ca
]
. However, PGI
or SNP do not promote the decay of InsP
elevated by
thrombin.
It is concluded that the cyclic nucleotides do not
modulate the Ca pumps and do not promote the
disappearance of InsP
; rather, they prevent InsP
from releasing Ca
from the stores. Such an
action of the cyclic nucleotides is unaffected by the specific PKC
inhibitor GF 109 203X, but it is prevented by staurosporine, which
inhibits both PKA and PKG.
The inhibition by the cyclic nucleotides
of the Ca-releasing action of InsP
is
observable directly in saponin-permeabilized platelets. The effect of
cAMP and cGMP are best evident at low InsP
concentrations
and disappear progressively with increasing InsP
. These
results are similar to those reported for cAMP in cerebellum-derived
microsomes(52) .
The inhibition by the cyclic nucleotides is
accompanied by the phosphorylation, operated by both PGI and SNP, of the InsP
receptor. The latter, as
evidenced by its reactivity with a specific antibody, appears as two
distinct bands in transblots from SDS-polyacrylamide gel
electrophoresis separations of human platelets. Both bands are
phosphorylated by PGI
and SNP, although the phosphorylation
is more evident on the heavier band. The presence of two bands may
indicate the occurrence in platelets of two different isoforms of the
receptor, as already described in several
organs(53, 54) . Alternatively, it may depend on
variable degrees of glycosylation (55) or it was the result of
a partial proteolysis at the NH
-terminal region of the
receptor.
Both PKA (54, 56, 57, 58) and PKG (59) are known to phosphorylate (at the same site) the
InsP receptor, which is also a substrate for PKC. In our
experiments, a phosphorylation by thrombin, sensitive to the specific
PKC inhibitor GF 109 203X is also observed; its onset is, however,
remarkably slower than that promoted by PGI
and SNP (see
also (54) ). The latter is fast enough to be compatible with
its intervention in the inhibition of the receptor function. The slower
PKC-dependent phosphorylation of the InsP
receptor may also
be involved in the control of its sensitivity to InsP
.
Indeed, we observed that staurosporine decreases Ca
reuptake into the stores after its release by thrombin (19 and
see also (60) ), and a decrease of Ca
reuptake was observed also in this study with the more specific
PKC inhibitor GF 109 203X.
Different and frequently contrasting
effects of the cAMP-dependent phosphorylation of the InsP receptor are reported in the recent literature. After the initial
observation by Supattapone et al.(52) that PKA
inhibits the InsP
-induced Ca
release in a
rat cerebellar microsomal fraction preloaded with Ca
,
a cAMP-dependent phosphorylation was reported to potentiate the
Ca
-releasing effect of InsP
in vesicles
reconstituted with a homotetrameric type I InsP
receptor (61) . In platelet membranes, data consistent with those of
Supattapone et al.(52) were reported in (44) , but the effect of cAMP was not appreciated in isolated
membranes(36) . This variability of data may relate to the
presence of regulatory factors in microsomes that regulate the
InsP
-dependent Ca
release. Besides, PKA
(PKG) may exert different effects on various subtypes of InsP
receptors that differ in their functional consequences. Indeed,
in some cells, such as the hepatocytes, the
Ca
-releasing action of agonists is potentiated rather
than inhibited by cAMP, which is therefore not expected to favor
Ca
reuptake in the stores(62, 63) .
In this study, we used intact human platelets to study the mechanism
by which PGI and SNP so powerfully depress the
[Ca
]
previously increased by
thrombin. By dissecting and studying separately each of the intervening
components, we could demonstrate that the action of cAMP and cGMP is to
inhibit the Ca
-releasing action of InsP
.
The effect is accompanied by the rapid phosphorylation of the receptor.
It seems inevitable to conclude that in the intact system the cyclic
nucleotides promote a receptor phosphorylation that prevents InsP
from releasing the store-associated Ca
.
It
may be concluded that both PGI and SNP, besides depressing
the agonist-induced activation of phospholipase C if they are presented
to the platelets prior to the agonist, as extensively demonstrated in
the literature, also oppose the effect of previously produced
InsP
, by specifically inhibiting its
Ca
-releasing property.