From the Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom
Received for publication, October 10, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Agonists elevate the cytosolic calcium
concentration in human platelets via a receptor-operated mechanism,
involving both Ca2+ release from intracellular stores
and subsequent Ca2+ entry, which can be inhibited by
platelet inhibitors, such as prostaglandin E1 and
nitroprusside which elevate cAMP and cGMP, respectively. In the present
study we investigated the mechanisms by which cAMP and cGMP modulate
store-mediated Ca2+ entry. Both prostaglandin
E1 and sodium nitroprusside inhibited thapsigargin-evoked
store-mediated Ca2+ entry and actin polymerization.
However, addition of these agents after induction of
store-mediated Ca2+ entry did not affect either
Ca2+ entry or actin polymerization. Furthermore,
prostaglandin E1 and sodium nitroprusside dramatically
inhibited the tyrosine phosphorylation induced by depletion of the
internal Ca2+ stores or agonist stimulation without
affecting the activation of Ras or the Ras-activated
phosphatidylinositol 3-kinase or extracellular signal-related
kinase (ERK) pathways. Inhibition of cyclic
nucleotide-dependent protein kinases prevented inhibition
of agonist-evoked Ca2+ release but it did not have any
effect on the inhibition of Ca2+ entry or actin
polymerization. Phenylarsine oxide and vanadate, inhibitors of
protein-tyrosine phosphatases prevented the inhibitory effects of the
cGMP and cAMP elevating agents on Ca2+ entry and actin
polymerization. These results suggest that Ca2+ entry in
human platelets is directly down-regulated by cGMP and cAMP by a
mechanism involving the inhibition of cytoskeletal reorganization via
the activation of protein tyrosine phosphatases.
In platelets and other nonexcitable cells, receptor stimulation
promotes the entry of extracellular Ca2+ through the plasma
membrane (1). A major mechanism for Ca2+ influx is
store-mediated Ca2+ entry
(SMCE),1 in which the filling
state of the internal Ca2+ stores regulates the entry of
Ca2+ (2). Although mechanisms involved in the activation of
this pathway are still not well understood, hypotheses have considered both indirect and direct coupling mechanisms. Indirect coupling assumes
the existence of a diffusible messenger generated by the Ca2+ storage organelles (3-6). The direct coupling
(conformational coupling) hypothesis proposes a physical interaction
between the endoplasmic reticulum and the plasma membrane (7).
Recently, a revised model for direct coupling, the secretion-like
coupling model, has been proposed to operate in several cell types (8), including human platelets (9). The secretion-like coupling model is
based on a reversible trafficking and coupling of the endoplasmic
reticulum with the plasma membrane that, in platelets, appears to
involve IP3 receptors in the endoplasmic reticulum and TRP1
channels in the plasma membrane (10).
In the secretion-like coupling model for SMCE, the actin cytoskeleton
plays an important role, which has been demonstrated in several cell
types (11, 12), including platelets (5, 9). Platelet activation is
accompanied by a dramatic increase in tyrosine phosphorylation of many
cellular proteins (13). A role for protein tyrosine phosphorylation in
the activation of SMCE has been reported on the basis of the
correlation between depletion of the intracellular Ca2+ and
increases in phosphotyrosine levels, as well as the effects of
different tyrosine kinase inhibitors on agonist and thapsigargin-evoked Ca2+ entry (6, 14-16). We have recently reported that the
effects of tyrosine kinases on SMCE are mediated through the
reorganization of the actin cytoskeleton in human platelets (17).
In contrast to platelet agonists, inhibitors of platelet activation,
which elevate the levels of cAMP or cGMP, antagonize agonist-evoked
increases in cytosolic calcium (1). The mechanisms of action of cAMP
and cGMP-elevating agents have been reported to involve the activation
of cAMP-dependent and cGMP-dependent protein
kinases (PKA and PKG), which in turn inhibit agonist-induced Ca2+ mobilization from the intracellular stores and hence
SMCE (18, 19). In the present study we sought to expand our
understanding of the mechanisms by which cAMP and cGMP-elevating
factors affect SMCE in platelets. We report here that prostaglandin
E1 (PGE1) and the nitrovasodilator, sodium
nitroprusside (SNP), which elevate the levels of cAMP and cGMP,
respectively, inhibit agonist-evoked Ca2+ release, an
action which is reversed by treatment with selective inhibitors of PKA
and PKG, respectively. Our findings also indicate that PGE1
and SNP inhibit both SMCE and actin polymerization by a mechanism
independent of PKA and PKG and which requires activation of
protein-tyrosine phosphatases. These results indicate that the
inhibition of agonist-evoked elevations in
[Ca2+]i by cAMP- or cGMP-elevating agents might
have two components: a reduction in Ca2+ store depletion
mediated by PKA or PKG, with a consequent reduction in SMCE, and a
direct effect on Ca2+ entry mediated by protein-tyrosine
phosphatases and inhibition of actin polymerization.
Materials--
Fura-2 acetoxymethyl ester (fura-2/AM) was from
Texas Fluorescence (Austin, TX). Apyrase (grade V), aspirin, bovine
serum albumin, paraformaldehyde, Nonidet P-40, fluorescein
isothiocyanate-labeled phalloidin, thrombin, ADP, sodium nitroprusside,
sodium vanadate, PGE1, and thapsigargin (TG) were
from Sigma (Poole, Dorset, United Kingdom). KT5823, KT5720, ionomycin,
and phenylarsine oxide (PAO) were from Calbiochem (Nottingham, UK).
Anti-VASP Ser239 monoclonal antibody was from Alexis Corp.
(Nottingham, UK). Pan-Ras (Ab-3) monoclonal antibody was from Oncogene
Science (Cambridge, MA). Phospho-p44/42 extracellular signal-regulated
kinases (ERK) monoclonal antibody (E10) was from New England Biolabs
(Beverly, MA). Anti-phosphotyrosine monoclonal antibody (4G10),
anti-phospho-Akt/PKB Platelet Preparation--
Fura-2-loaded platelets were prepared
as described previously (5). Briefly, blood was obtained from healthy
volunteers and mixed with one-sixth volume of acid/citrate dextrose
anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid, and 111 D-glucose. Platelet-rich plasma was
then prepared by centrifugation for 5 min at 700 × g
and aspirin (100 µM) and apyrase (40 µg/ml) added.
Platelet-rich plasma was incubated at 37 °C with 2 µM
fura-2/AM for 45 min. For loading with dimethyl-BAPTA, cells were
incubated for 30 min at 37 °C with 10 µM
dimethyl-BAPTA AM. Cells were then collected by centrifugation at
350 × g for 20 min and resuspended in HEPES-buffered
saline containing (in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4, pH 7.45, and
supplemented with 0.1% (w/v) bovine serum albumin and 40 µg/ml apyrase.
Measurement of Intracellular Free Calcium Concentration
([Ca2+]i)--
Fluorescence was recorded
from 1.5-ml aliquots of magnetically stirred platelet suspension
(108 cells/ml) at 37 °C using a Cairn Research
Spectrophotometer (Cairn Research Ltd., Sittingbourne, Kent, UK) with
excitation wavelengths of 340 and 380 nm and emission at 500 nm.
Changes in [Ca2+]i were monitored using the
fura-2 340/380 fluorescence ratio and calibrated according to the
method of Grynkiewicz et al. (20).
Determination of Ca2+ Entry--
Ca2+
influx in platelets that had been store depleted using TG was estimated
using the integral of the rise in [Ca2+]i for
21/2 min after addition of CaCl2 (5). When platelets were preincubated with inhibitors, Ca2+ entry was corrected
by subtraction of the rise in [Ca2+]i due to
leakage of the indicator. TG-induced Ca2+ release was
estimated using the integral of the rise in
[Ca2+]i for 3 min after its addition.
Thrombin-evoked Ca2+ elevation or release was measured as
the integral of the rise in [Ca2+]i above basal
for 90 s after the addition of the agonist in the presence of
external Ca2+ or in a Ca2+-free medium (with
100 µM EGTA), respectively.
Measurement of F-actin Content--
The F-actin content of
resting and activated platelets was determined according to a
previously published procedure (5). Briefly, washed platelets (2 × 108 cells/ml) were activated in HEPES-buffered saline.
Samples of platelet suspension (200 µl) were transferred to 200 µl
of ice-cold 3% (w/v) formaldehyde in phosphate-buffered saline (PBS)
for 10 min. Fixed platelets were permeabilized by incubation for 10 min with 0.025% (v/v) Nonidet P-40 detergent dissolved in PBS. Platelets were then incubated for 30 min with fluorescein isothiocyanate-labeled phalloidin (1 µM) in PBS supplemented with 0.5% (w/v)
bovine serum albumin. After incubation the platelets were collected by
centrifugation in an MSE Micro-Centaur Centrifuge (MSE Scientific
Instruments, Crawley, Sussex, UK) for 60 s at 3000 × g and resuspended in PBS. Staining of 2 × 107 cells/ml was measured using a PerkinElmer Fluorescence
Spectrophotometer (PerkinElmer Life Sciences, Norwalk, CT). Samples
were excited at 496 nm and emission was at 516 nm.
Protein Tyrosine Phosphorylation--
Protein tyrosine
phosphorylation was detected by gel electrophoresis and Western
blotting (6). Platelets stimulation was terminated by mixing with an
equal volume of 2 × Laemmli's buffer (21) with 10%
dithiothreitol followed by heating for 5 min at 95 °C.
One-dimensional SDS electrophoresis was performed with 10%
polyacrylamide minigels and separated proteins were electrophoretically transferred, for 2 h at 0.8 mA/cm2, in a semi-dry
blotter (Hoefer Scientific, Newcastle, Staffs., UK) onto nitrocellulose
for subsequent probing. Blots were incubated overnight with 10% (w/v)
bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBST)
to block residual protein-binding sites. Immunodetection of tyrosine
phosphorylation was achieved using the anti-phosphotyrosine antibody
4G10 diluted 1:2500 in TBST for 1 h. The primary antibody was
removed and blots washed six times for 5 min each with TBST. To detect
the primary antibody, blots were incubated with horseradish
peroxidase-conjugated ovine anti-mouse IgG antibody diluted 1:10000 in
TBST, washed six times in TBST, and exposed to enhanced
chemiluminescence reagents for 1 min. Blots were then exposed to
preflashed photographic film. Densitometric measurements were made
using a Quantimet 500 densitometer (Leica, Milton Keynes, UK).
Quantitative analysis of VASP Ser239
Phosphorylation--
Phosphorylation of the residue Ser239
of VASP, a phosphorylation site preferred by PKG but also used by PKA
(22), was measured by SDS-PAGE and Western blotting as described above
but using a specific anti-VASP Ser239 monoclonal antibody
(16C2) diluted 1:1000 in TBST as previously described (22).
Analysis of Akt/PKB Analysis of Diphosphorylated p42/p44 ERK--
Analysis of
diphosphorylated p42/p44 ERK was performed by SDS-PAGE and Western
blotting as described above but using a specific phospho-p44/42 MAP
kinase monoclonal antibody (E10) diluted 1:1500 in TBST as previously
described (24).
Subcellular Fractionation--
Human platelet fractionation was
carried out according to a previously published procedure (5). Briefly,
platelets were pelleted in a microcentrifuge and the pellets were
quickly resuspended in 0.5 ml of ice-cold Tris-HCl buffer containing:
10 mM Tris-HCl (pH 7.2), 158 mM NaCl, 1 mM EGTA, 50 µg/ml leupeptin, 5 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. The suspensions were
sonicated and intact platelets were removed by centrifugation at
1,500 × g. The platelet lysate was centrifuged at
100,000 × g at 4 °C for 60 min to obtain membrane
and cytosolic fractions. Membranes were washed with PBS with 1 mM Na3VO4 at 4 °C and
resuspended in Tris-HCl buffer containing: 10 mM Tris-HCl,
pH 7.2, 158 mM NaCl, 1 mM EGTA, 0.1% SDS, 1%
sodium deoxycholate, 1% Triton X-100, 50 µg/ml leupeptin, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. Lysates
were centrifuged at 16,000 × g for 5 min to remove
insoluble substances. Lysates of the subcellular fractions or total
cell lysates (50 µg/well) were analyzed by Western blotting with
pan-Ras (Ab-3) monoclonal antibody diluted 1:500 in TBST. To detect the
primary antibody, blots were incubated with horseradish
peroxidase-conjugated anti-mouse IgG antibody diluted 1:10,000 in TBST
and exposed to enhanced chemiluminescence reagents for 1 min as
described above. Blots were then exposed to preflashed photographic film.
Statistical Analysis--
Analysis of statistical significance
was performed using Student's t test. For multiple
comparisons, one-way analysis of variance combined with the Dunnett
test was used.
Regulation of Agonist-induced Ca2+ Mobilization by
PGE1 and SNP--
Treatment of fura-2-loaded human
platelets with the physiological agonists thrombin (0.1 unit/ml) or ADP
(40 µM) evoked rises in [Ca2+]i in
both a medium containing 1 mM Ca2+ and in the
absence of external Ca2+ (100 µM EGTA was
added). In agreement with previous studies (18, 19) thrombin- and
ADP-induced Ca2+ elevations in human platelets were
significantly inhibited when the cells were preincubated for 1 min with
5 µM PGE1 or 100 µM SNP (Fig.
1; p < 0.01;
n = 6-11). In contrast, PGE1 and SNP had no effect on resting [Ca2+]i (Fig. 1).
Effect of PGE1 and SNP on Store-mediated
Ca2+ Entry in Platelets--
In a Ca2+-free
medium, TG, a specific inhibitor of the endomembrane
Ca2+-ATPase (SERCA, Ref. 25), evoked a sustained elevation
in [Ca2+]i due to the release of Ca2+
from internal stores. The subsequent addition of Ca2+
(final concentration 300 µM) to the external medium
induced a rapid increase in [Ca2+]i indicative of
SMCE (Fig. 2). Treatment of platelets for
1 min with 5 µM PGE1 or 100 µM
SNP decreased Ca2+ entry to 56.2 ± 4 and 52.5 ± 7.2% of control (vehicle added; Fig. 2; p < 0.001;
n = 6-9). Platelets preincubated for 1 min with 5 µM PGE1 or 100 µM SNP showed an
identical release of Ca2+ from the intracellular stores
after treatment with TG in comparison with untreated cells (99.04 ± 1.9 and 98.8 ± 2.6% of control after treatment with
PGE1 or SNP, respectively).
Regulation of Actin Polymerization by PGE1 or SNP in
Human Platelets--
Recently a role for the actin cytoskeleton in the
activation of SMCE has been suggested in several cells types (11, 12, 26). As we have previously shown (17), TG induces actin polymerization in the absence of a detectable rise in [Ca2+]i in
platelets loaded with the Ca2+ chelator dimethyl-BAPTA.
After dimethyl-BAPTA loading, treatment of platelets with 1 µM TG in a Ca2+-free medium raised the
F-actin content by 29.7 ± 6.4% compared with control
unstimulated cells. Treatment with various concentrations of
PGE1 or SNP did not significantly modify the F-actin
content of resting platelets; however, TG-induced actin polymerization was inhibited in a concentration-dependent manner, with
IC50 values of 53.0 ± 2.9 nM and 1.7 ± 0.4 µM, respectively, and complete inhibition at 5 and
100 µM, respectively (Fig.
3; p < 0.05;
n = 6).
Stimulation of dimethyl-BAPTA-loaded human platelets with thrombin (0.1 unit/ml) or ADP (40 µM) for 3 min increased F-actin content by 32.1 ± 3.2% and 23.6 ± 2.1% of control,
respectively. Consistent with the above, treatment of platelets with 5 µM PGE1 or 100 µM SNP abolished
both thrombin- and ADP-induced actin polymerization (data not shown).
Effect of PGE1 and SNP on the Maintenance of
Store-mediated Ca2+ Entry--
We have previously reported
that the integrity of the actin cytoskeleton is essential for both the
activation and maintenance of SMCE in platelets (9). To investigate the
effects of cAMP and cGMP on the maintenance of SMCE we examined the
effect of PGE1 and SNP, respectively, on Ca2+
entry in platelets after SMCE had been previously stimulated using
TG.
Fig. 4 shows the effect of adding
PGE1 or SNP to store-depleted human platelets. 5 µM PGE1 (Fig. 4A), 100 µM SNP (Fig. 4B), or the vehicles were added 3 min after TG and cells were then incubated for a further 1 min before
the addition of Ca2+ to the medium (final concentration 300 µM) to initiate Ca2+ entry. At the time when
PGE1 and SNP were added Ca2+ entry was already
stimulated (data not shown, but see Fig. 2). Addition of
PGE1 or SNP after Ca2+ store depletion did not
significantly modify subsequent Ca2+ entry (Fig. 4;
p = 0.87; n = 6). Consistent with these
results, treatment of human platelets for 1 min with 5 µM
PGE1 or 100 µM SNP after SMCE had been
activated by the addition of TG did not alter TG-evoked increase in the
F-actin content (Table I;
n = 6). These findings show that cAMP and cGMP inhibit
the activation but not the maintenance of SMCE. In addition, these
observations indicate that PGE1 and SNP do not act either
as Ca2+ channel blockers or Ca2+ chelators or
as nonspecific inhibitors of actin polymerization.
Role of PKA and PKG in the Inhibitory Effects of PGE1
and SNP on Actin Polymerization--
To investigate the possible roles
of PKA and PKG in the responses observed after treatment with
PGE1 and SNP we used KT5720 and KT5823, highly specific,
cell-permeant inhibitors of PKA and PKG activity, respectively (27).
Since VASP has been reported to be a substrate for both PKA and PKG
(22, 28) and the residue Ser239 is the site preferred by
PKG is also used by PKA in platelets (29, 30), we monitored PKA and PKG
activity by testing the effect of the inhibitors on PGE1-
and SNP-induced phosphorylation of this residue. As shown in Fig.
5, treatment of platelets for 30 min with
KT5720 or KT5823 inhibited phosphorylation of VASP at residue
Ser239 in a concentration-dependent manner,
with IC50 values of 520.0 ± 17.4 and 168.8 ± 14.1 nM, respectively, and complete inhibition at 3 and 1 µM, respectively (Fig. 5; p < 0.001;
n = 4).
Treatment of human platelets for 30 min with 3 µM KT5720
or 1 µM KT5823 did not alter the F-actin content of
unstimulated platelets. In addition, these treatments did not reverse
the inhibitory effects of PGE1 or SNP, respectively, on
actin polymerization (Table II;
n = 6). These results indicate that the effects of PGE1 and SNP are independent of PKA and PKG,
respectively.
PGE1 and SNP Prevent Protein Tyrosine Phosphorylation
in Human Platelets--
Agonist- and store depletion-induced protein
tyrosine phosphorylation was assessed by gel electrophoresis and
Western blotting with a specific antiphosphotyrosine antibody.
Platelets heavily loaded with the Ca2+ chelator
dimethyl-BAPTA were used for this study so as to eliminate Ca2+-dependent tyrosine phosphorylation
(31).
Dimethyl-BAPTA-loaded platelets were incubated for 1 min at 37 °C
with 5 µM PGE1 or 100 µM SNP or
the vehicle and Ca2+ stores were depleted using TG (1 µM). Samples for protein phosphorylation analysis were
taken from the spectrophotometer cuvette 10 s prior the addition
of PGE1, SNP, or the vehicle and 10 s before and 10 and 180 s after the addition of TG. As shown in Fig.
6, A and B,
pretreatment with PGE1 or SNP significantly reduced protein tyrosine phosphorylation relative to its control in store depleted cells (e.g. bands at 72 and 130 kDa; n = 4).
Similar results were observed when cells were stimulated with the
physiological agonists thrombin (0.1 unit/ml; Fig. 6C and
D; n = 4) and ADP (40 µM; not shown). In addition, treatment with 5 µM PGE1
or 100 µM SNP significantly reduced tyrosine
phosphorylation in unstimulated platelets by 20 ± 5 and 25 ± 5%, respectively (p < 0.01; n = 4).
Effect of Protein-tyrosine Phosphatases on PGE1- and
SNP-induced Responses--
The possibility that the inhibitory effects
observed after treatment with PGE1 or SNP might be mediated
by protein-tyrosine phosphatases was tested by examining the effect of
the tyrosine phosphatase inhibitors PAO and vanadate. Under our
conditions, treatment of dimethyl-BAPTA-loaded human platelets for 30 min with either 10 µM PAO or 100 µM
vanadate increased the phosphotyrosine level of both resting and
stimulated cells without impairing TG-induced increase in the
phosphotyrosine level (Fig. 7;
n = 4). Interestingly, preincubation of platelets with
10 µM PAO or 100 µM vanadate for 30 min
completely reversed the inhibition of actin polymerization observed
after treatment with PGE1 or SNP (Table II;
n = 6), while this treatment per se did not
modify either the F-actin content of unstimulated cells or TG-induced
actin polymerization (data not shown).
Next we tested whether the inhibition of SMCE could be mediated by the
activation of tyrosine phosphatase activity. Cells treated with PAO or
vanadate showed similar resting levels of [Ca2+]i
to control platelets (data not shown). In addition, PAO or
vanadate-treated cells retained their ability to respond to
Ca2+ mobilizing agents, such as TG, which indicates that
this treatment did not affect the ability of platelets to store
Ca2+ in intracellular compartments as well as demonstrating
that these agents per se do not mobilize Ca2+
from the stores in platelets (data not shown). Platelets previously preincubated for 30 min with 10 µM PAO or 100 µM vanadate were treated for 1 min with PGE1,
SNP, or the vehicles and then stimulated with TG (200 nM)
in a Ca2+-free medium and 3 min later CaCl2
(final concentration 300 µM) was added. As shown in Table
III, incubation with protein-tyrosine phosphatase inhibitors almost completely reversed the inhibitory effect
of PGE1 and SNP on SMCE. In contrast, PGE1 and
SNP inhibited SMCE to the same extent in control cells or in platelets
preincubated for 30 min with the PKA and PKG inhibitors, KT5720 (3 µM) and KT5823 (1 µM), respectively (Table
III; n = 6-10). These findings are consistent with the
role of protein-tyrosine phosphatases in actin filament polymerization
and indicate that the effect exerted by cyclic nucleotides on SMCE is
independent of PKA and PKG.
The effect of PGE1 and SNP on thrombin-evoked
Ca2+ mobilization is shown in Table
IV. The effects of either
PGE1 (5 µM) or SNP (100 µM) on
thrombin-evoked Ca2+ release were almost completely
reversed by pretreatment for 30 min with 3 µM KT5720 or 1 µM KT5823, respectively, in contrast, 10 µM
PAO or 100 µM vanadate had negligible effects (Table IV; n = 6). Pretreatment of platelets with KT5720 or KT5823
partially reversed the effect of PGE1 or SNP on
thrombin-induced Ca2+ responses in a medium containing 1 mM Ca2+ (Table IV; p < 0.05;
n = 6). Similar results were obtained when platelets
were treated with PAO or vanadate (Table IV; p < 0.05; n = 6). More importantly, the combined effect of
inhibitors of either PKA or PKG and of tyrosine phosphatases completely
prevented the inhibition induced by PGE1 and SNP on
thrombin-induced Ca2+ elevations in the presence of 1 mM external Ca2+ (Table IV; p < 0.001; n = 6).
Role of the Ras-dependent Pathways on PGE1-
and SNP-induced Responses--
The activity of Ras proteins has been
shown to be required for the activation of SMCE in several cell types
(32, 33) including platelets (5). To investigate whether
PGE1 and SNP impair the activation of Ras we have examined
their effects on the association of Ras with membranes, a process
essential for Ras activation (34). In resting platelets, pan-Ras
immunoreactive proteins were detected in both cytosolic and membrane
fractions (Fig. 8A). As
reported previously (5), TG induces translocation of Ras to the
membrane fraction, an effect that was unmodified when platelets were
incubated for 1 min with either PGE1 or SNP (Fig.
8A, n = 3), treatments which also did not
alter the distribution of Ras in resting cells (data not shown). We
also investigated the effect of PGE1 and SNP on the
activation of phosphatidylinositol 3-kinase (PI3K) and ERK, which have
been proposed as candidates for the cytoskeleton-dependent
and -independent branches of the Ras-dependent activation
of SMCE in platelets, respectively
(35).2 PI3K activity was
investigated by examining the phosphorylation of its substrate,
Akt/PKB Regulation of Plasma Membrane Ca2+-ATPase Activity by
SNP--
Previous studies have shown that increasing platelet
cytosolic cAMP with PGE1 or forskolin stimulates
plasma-membrane Ca2+-ATPase (PMCA) activity in these cells
(31). Using a previously published protocol (37) we have investigated
the possible effect of cGMP on PMCA activity. In a
Ca2+-free medium (100 µM EGTA added),
inhibition of SERCA using 1 µM TG in the presence of a
low concentration of ionomycin (50 nM; required for
extensive depletion of the intracellular Ca2+ stores in
platelets), resulted in a transient increase in
[Ca2+]i due to release of Ca2+ from
intracellular stores. Since under these conditions the exclusive mechanism for Ca2+ extrusion in human platelets is the PMCA
(37), we estimated PMCA activity by examining the rate of decay (see
Ref. 37). Fig. 9A shows the
effect of preincubation for 1 min with 100 µM SNP on the
response evoked by TG (1 µM) plus ionomycin (50 nM). Preincubation with SNP did not modify the release of
Ca2+ from the intracellular stores stimulated by TG plus
ionomycin; however, the rate of decay was enhanced by 135%. The decay
constants were 0.0150 ± 0.0008 in SNP-treated platelets and
0.0114 ± 0.0009 in paired controls (n = 6;
p < 0.01). The effect of SNP was essentially abolished
by preincubation with the PKG inhibitor KT5823. The decay constants
were 0.0101 ± 0.0006 in cells preincubated for 30 min with 1 µM KT5823 and then treated with SNP and 0.0105 ± 0.0006 in controls (Fig. 9B; n = 6;
p = 0.30).
Ca2+ mobilization in nonexcitable cells regulates such
diverse processes as secretion, contraction, gene expression, and
apoptosis. In human platelets, most agonists elevate
[Ca2+]i through receptor-dependent
mechanisms, activating both the release of Ca2+ from the
intracellular stores and Ca2+ entry across the plasma
membrane. These responses are antagonized by cAMP- and cGMP-elevating
agents. Agents such as PGE1 or iloprost stimulate cAMP
formation. On the other hand, nitrovasodilators, such as nitroprusside,
are potent cGMP-elevating platelet inhibitors (1).
Several studies in platelets have reported that the effects of cyclic
nucleotides on the Ca2+ entry process are secondary to
their inhibitory effects on intracellular Ca2+ release (18,
19, 38). In the present study we shed new light on the mechanism of
action of cyclic nucleotides on SMCE. By reducing the preincubation
time with the cAMP- and cGMP-elevating agents we were able to detect
for the first time substantial inhibition of SMCE without the
modification of TG-induced release. These findings strongly suggest
that cyclic nucleotides exert a negative regulation of Ca2+
entry independently of the extent of release from intracellular Ca2+ stores.
Recently it has been reported that the integrity of the actin
cytoskeleton is required for the activation and maintenance of SMCE in
different cell types (11, 12), including platelets (5, 9). Hence, we
have investigated whether cyclic nucleotides interfere with actin
polymerization. Treatment of platelets with the cAMP- and
cGMP-elevating agents, PGE1 and SNP, inhibited actin polymerization stimulated by TG or the physiological agonists ADP and
thrombin. This inhibition of actin polymerization was observed in cells
heavily loaded with the Ca2+ chelator dimethyl-BAPTA, and
thus was independent of any effects of the cyclic nucleotides on
[Ca2+]i. These results, together with the degree
of inhibition of SMCE, are consistent with the effects of the
inhibitors of actin polymerization cytochalasin D and latrunculin A on
SMCE in human platelets (5, 9). These agents inhibited SMCE by 50% at
concentrations that abolished actin polymerization in platelets. Therefore, these findings suggest that the effect of cyclic nucleotides on SMCE might be mediated by impairment of actin filament polymerization.
Previous studies have shown that the effects of cyclic nucleotides are
not mediated by acceleration of Ca2+ removal from the
cytosol (38). Since more recent studies have shown that cAMP stimulates
PMCA activity (31), we performed a series of studies to test the
effects of cGMP on Ca2+ extrusion. We have reported that
the main mechanism for Ca2+ extrusion in human platelets is
the PMCA (37, 39). Our findings demonstrate that, as well as
cAMP-elevating agents (31), SNP significantly increases
Ca2+ extrusion in platelets, a process that is entirely
mediated by the PKG, since inhibition of this kinase abolished the
effect of SNP. To evaluate whether Ca2+ entry is directly
down-regulated by the cyclic nucleotides or if their effects are
mediated by acceleration of Ca2+ extrusion we added
PGE1 or SNP to the platelet suspension once store-mediated
Ca2+ entry had been preactivated using TG. Our results show
that treatment with cAMP- and cGMP-elevating agents after the
activation of SMCE has no effect on Ca2+ entry, while PMCA
activity is increased. These observations indicate that, in agreement
with previous studies (38), cyclic nucleotides do not inhibit
elevations in [Ca2+]i by accelerating
Ca2+ extrusion alone, but that cAMP and cGMP are also
involved in regulating the activation but not the maintenance of SMCE
in platelets. Consistent with this, neither PGE1 nor SNP
impaired actin polymerization in preactivated platelets.
Most cellular functions exerted by cyclic nucleotides are mediated by
the activation of cAMP-dependent and
cGMP-dependent protein kinases. To determine whether PKA
and PKG mediate these responses we used KT5720 and KT5823, selective
cell-permeant inhibitors of PKA and PKG, respectively (27). By
monitoring PKA and PKG activity we found that treatment of platelets
for 1 min with 5 µM PGE1 or 100 µM SNP effectively activated PKA or PKG activity, respectively. Our present results and numerous previous studies with
cAMP- and cGMP-elevating agents (for review, see Refs. 1, 18, and 19)
suggest that inhibition of agonist evoked intracellular Ca2+ discharge is an effect mediated by both PKA and PKG.
Although phosphorylation of VASP correlates with the inhibition of
Ca2+ release, at present we do not have evidence for the
involvement of VASP in this process. However, a recent report has shown
that VASP is not required for the inhibition of Ca2+
mobilization in murine platelets mediated by cAMP- and cGMP-elevating agents (28). In contrast, the analysis of the F-actin content and SMCE
clearly excluded PKA and PKG as candidate proteins involved in cyclic
nucleotide-mediated regulation of actin polymerization and
Ca2+ entry, since these processes were unaffected by KT5720
or KT5823. The partial recovery observed in thrombin-induced
Ca2+ elevation in the presence of 1 mM external
Ca2+ after incubation with KT5720 or KT5823 can be
explained by the effect of these inhibitors on PGE1- and
SNP-induced reductions in Ca2+ release. Different results
have been reported in vascular endothelial cells where cGMP inhibits
SMCE via a PKG-dependent mechanism (40).
An alternative pathway, responsible for the inhibitory effects of cAMP-
and cGMP-elevating agents on Ca2+ entry and actin
polymerization, remains to be identified. Platelet activation is
accompanied by a dramatic increase in tyrosine phosphorylation of many
cellular proteins (13). A role for protein tyrosine phosphorylation in
the regulation of SMCE and actin polymerization has been proposed on
the basis of the correlation between increases in phosphotyrosine
levels and the filling state of the Ca2+ stores, as well as
the effects of different tyrosine kinase inhibitors on agonist and
thapsigargin-evoked Ca2+ entry and actin polymerization
(14-17). In agreement with previous studies (38, 41), we have found
that cAMP- and cGMP-elevating agents inhibit agonist- or TG-stimulated
tyrosine phosphorylation in dimethyl-BAPTA-loaded platelets. Therefore,
we investigated whether protein-tyrosine phosphatases might be involved
in the inhibitory effects of cAMP- and cGMP-elevating agents. Treatment of platelets with the protein-tyrosine phosphatase inhibitors, PAO and
vanadate, prevented the effect of both PGE1 and SNP on actin polymerization and SMCE. We have found that PAO and vanadate do
not impair agonist-evoked increases in phosphotyrosine levels. In
addition, this treatment per se did not alter either basal or stimulated F-actin content or SMCE. These data suggest that the
inhibitory effects of cAMP- and cGMP-elevating agents on actin polymerization and SMCE are mediated by the activation of tyrosine phosphatases. These findings are in agreement with earlier reports showing a role for tyrosine kinases in actin reorganization (17) and
SMCE (14-16). Consistent with a recent study providing evidence that
suggests that tyrosine kinases and Ras proteins have independent effects in the activation of SMCE in human platelets (17), we have
found that treatment with PGE1 or SNP did not interfere
with Ras activation or the Ras-activated PI3K or ERK pathways. The effect of tyrosine phosphatases on cAMP and cGMP-induced inhibitions of
agonist-evoked Ca2+ mobilization is more complex. Our
results indicate that tyrosine phosphatases are not involved in the
effects of cyclic nucleotides on Ca2+ release from the
intracellular stores, which appear to be mediated entirely through PKA
and PKG. However, the effects of cAMP and cGMP on agonist-evoked
elevations in [Ca2+]i as a whole (which involves
Ca2+ release and entry) are dependent on both cyclic
nucleotide-dependent kinases, which regulate
Ca2+ release, and tyrosine phosphatases, which are involved
in the inhibition of Ca2+ entry. Thus, the effects of cAMP-
and cGMP-elevating agents, which are partially impaired by the
inhibition of either of these pathways, are abolished by the inhibition
of both.
In conclusion, we report here for the first time that SMCE in
human platelets is directly down-regulated by cAMP and cGMP by a
mechanism involving the activation of protein-tyrosine phosphatases. Therefore, two different cyclic nucleotide-dependent
mechanisms operate during the regulation of agonist-induced
Ca2+ elevation: a PKA-/PKG- dependent mechanism for the
inhibition of Ca2+ release and a PKA-/PKG-independent
tyrosine phosphatase-dependent mechanism for the direct
inhibition of Ca2+ entry. The findings that cAMP and
cGMP-elevating agents prevent the activation of SMCE, but not its
maintenance, via the activation of tyrosine phosphatases is consistent
with earlier observations indicating a role for tyrosine kinases in the
activation but not in the maintenance of SMCE (17).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Ser473) monoclonal antibody, and
horseradish peroxidase-conjugated rabbit anti-sheep IgG antibody were
from Upstate Biotechnology (Lake Placid, NY). Horseradish
peroxidase-conjugated ovine anti-mouse IgG antibody (NA931) was from
Amersham Pharmacia Biotech (Little Chalfont, Bucks., UK).
Dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester was from Molecular Probes (Leiden, The
Netherlands). All other reagents were of analytical grade.
Ser473
Phosphorylation--
Phosphorylation of Akt/PKB
on the residue
Ser473 was assessed by SDS-PAGE and Western blotting as
described above but using a specific anti-phospho-Akt/PKB
(Ser473) monoclonal antibody diluted 1:1500 in TBST as
previously described (23), followed by incubation with horseradish
peroxidase-conjugated rabbit anti-sheep IgG antibody diluted 1:10000 in TBST.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of PGE1 or SNP on
thrombin- or ADP-induced elevations in [Ca2+]i in
platelets. Fura-2-loaded human platelets were incubated at
37 °C for 1 min in the presence of 5 µM
PGE1, 100 µM SNP, or the vehicles
(Control). At the time of the experiment 1 mM
Ca2+ or 100 µM EGTA was added as indicated.
Cells were then stimulated with 0.1 unit/ml thrombin (A-D)
or 40 µM ADP (E-F). Elevations in
[Ca2+]i were monitored using the 340/380 nm ratio
as described under "Experimental Procedures." Traces shown are
representative of 6 to 11 independent experiments.
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[in a new window]
Fig. 2.
Effects of PGE1 or SNP on
TG-induced SMCE. Fura-2-loaded human platelets were incubated at
37 °C in the presence of 5 µM PGE1
(A), 100 µM SNP (B), or the
vehicles (Control). At the time of the experiment 100 µM EGTA was added. Cells were then stimulated with TG
(200 nM) and 3 min later CaCl2 (final
concentration, 300 µM) was added to the medium to
initiate Ca2+ entry. Elevations in
[Ca2+]i were monitored using the 340/380 nm ratio
as described under "Experimental Procedures." Traces are
representative of six to nine independent experiments.
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Fig. 3.
Effect of PGE1 or SNP on the
actin filament content of TG-stimulated human platelets.
Dimethyl-BAPTA-loaded human platelets were incubated for 1 min in the
absence or presence of various concentrations of PGE1
(A) or SNP (B) as indicated. Platelets were then
stimulated with 200 nM TG. Samples were removed 5 s
before and 3 min after adding TG and actin filament content was
determined as described under "Experimental Procedures." Values
given are TG-induced actin-filament formation as a percentage of
control (TG-stimulated nontreated cells) and results are expressed as
mean ± S.E. of six separate determinations. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, compared with control.
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Fig. 4.
Effect of PGE1 or SNP on the
maintenance of SMCE. Fura-2-loaded human platelets were suspended
in a Ca2+-free medium (100 µM EGTA added) as
described under "Experimental Procedures." Cells were then
stimulated with TG (200 nM) and, 3 min later, 5 µM PGE1, 100 µM SNP, or the
vehicles (Control) were added, as indicated by the
arrow. CaCl2 (final concentration 300 µM) was added to the medium 1 min after SNP,
PGE1, or the vehicles to initiate Ca2+ entry.
Elevations in [Ca2+]i were monitored using the
340/380 nm ratio as described under "Experimental Procedures."
Traces are representative of six independent experiments.
HBS, HEPES-buffered saline.
Effects of PGE1 or SNP on the F-actin content of platelets in
which store-mediated calcium entry had been preactivated
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Fig. 5.
Quantitative analysis of Ser239
phosphorylated VASP in platelets incubated with KT5720 or KT5823.
Human platelets were incubated with various concentrations of KT5720
(left panel), KT5823 (right panel), or the
vehicles. Cells were stimulated for 1 min with 5 µM
PGE1 or 100 µM SNP as indicated and then
lysed. Samples were subjected to SDS-PAGE and analyzed by Western
blotting with the specific monoclonal antibody to Ser239
phosphorylated VASP as described under "Experimental Procedures."
Bands were revealed using chemiluminescence and quantified using
scanning densitometry. The upper panels show VASP
Ser239 phosphorylation results from a representative
experiment of at least three others. The bottom panels show
the quantification of VASP Ser239 phosphorylated. Values
are mean ± S.E. (n = 4) expressed as the
percentage of the increase evoked by 5 µM
PGE1 (A) or 100 µM SNP
(B) above control unstimulated values.
Effects of PKA, PKG, and protein-tyrosine phosphatases on actin
polymerization in platelets
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Fig. 6.
Effect of PGE1 or SNP on TG- or
thrombin-evoked protein tyrosine phosphorylation.
Dimethyl-BAPTA-loaded platelets were incubated for 1 min with 5 µM PGE1 (A and C,
lanes 5-8), 100 µM SNP (B and
D; lanes 5-8), or the vehicles as control
(lanes 1-4) and then stimulated with TG (1 µM) or thrombin (0.1 unit/ml). At the times indicated
100-µl aliquots were removed and the reaction terminated by mixing
with an equal volume of 2 × Laemmli's buffer with 10%
dithiothreitol. Proteins were analyzed by SDS-PAGE and subsequent
Western blotting with a specific antiphosphotyrosine antibody as
described under "Experimental Procedures." Molecular size is
indicated on the right. The panels show results from an
experiment representative of three others.
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Fig. 7.
Effect of PAO or vanadate on TG-stimulated
protein tyrosine phosphorylation. Dimethyl-BAPTA-loaded platelets
were incubated for 30 min with 10 µM PAO, 100 µM vanadate, or the vehicles (Control) and then
stimulated with TG (1 µM). At the times indicated
100-µl aliquots were removed and the reaction terminated by mixing
with an equal volume of 2 × Laemmli's buffer with 10%
dithiothreitol. Proteins were analyzed by SDS-PAGE and subsequent
Western blotting with a specific antiphosphotyrosine antibody as
described under "Experimental Procedures." Molecular size is
indicated on the right. The panels show results from a
representative experiment of three others.
Effects of PKA, PKG, or protein-tyrosine phosphatases on TG-stimulated
store-mediated Ca2+ entry
Effects of PKA, PKG, or protein-tyrosine phosphatases on
thrombin-stimulated Ca2+ elevations in the presence or absence
of external Ca2+
on Ser473. It is well known that activation of
PI3K results in phosphorylation of Akt/PKB
on both
Ser473 and Thr308 (36). As shown in Fig.
8B, treatment of platelets with TG (200 nM)
resulted in an increase in phosphorylation of Akt/PKB
on Ser473 by 4.7 ± 1.2-fold. This increase was abolished
by preincubation for 30 min with 10 µM LY 294002, a
concentration that selectively inhibits PI3K activity (35) (Fig.
8B; n = 3). In contrast, treatment for 1 min
with PGE1 or SNP did not modify either basal or TG-induced phosphorylation of Akt/PKB
on Ser473, indicating that
these agents do not affect PI3K activity. Similar results were obtained
when the effect of these agents on TG-induced ERK activation was
investigated. ERK activation was determined using an antibody that
specifically recognizes diphosphorylated and thus activated p42/44 ERK
as previously described (24). We have recently found that TG induces
ERK activation in human platelets, an action which is downstream of Ras
activation.2 As shown in Fig. 8C, treatment of
platelets for 1 min with PGE1 or SNP did not alter either
basal or TG-induced ERK activation (n = 3). Together,
these results indicate that PGE1- or SNP-induced inhibition
of Ca2+ entry is independent on the Ras-mediated activation
of SMCE in human platelets.
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Fig. 8.
Effects of PGE1 or SNP on
TG-induced membrane association of Ras and the activation of PI
3-kinase and ERK. A, dimethyl-BAPTA-loaded human
platelets were incubated for 1 min with 5 µM
PGE1, 100 µM SNP, or the vehicles as
indicated and then stimulated with TG (200 nM) in a
Ca2+-free medium. Samples were taken 5 s before
(lanes 1 and 2) and 3 min after adding TG
(lanes 3-8). Cytosolic fractions (C) and
membrane fractions (M) were isolated as described under
"Experimental Procedures." Lysates of the subcellular fractions
were analyzed by Western blotting with a pan-Ras antibody.
B, dimethyl-BAPTA-loaded platelets were incubated in the
absence or presence of either 10 µM LY294002 for 30 min
or 5 µM PGE1 or 100 µM SNP for
1 min as indicated and then stimulated with TG (200 nM) in
a Ca2+-free medium. Samples were taken 5 s before and
3 min after adding TG. Samples were subjected to SDS-PAGE and analyzed
by Western blotting with the specific monoclonal antibody to
Ser473 phosphorylated Akt/PKB as described under
"Experimental Procedures." C, dimethyl-BAPTA-loaded
human platelets were incubated for 1 min with 5 µM
PGE1, 100 µM SNP, or the vehicles as
indicated and then stimulated with TG (200 nM) in a
Ca2+-free medium. Samples were taken 5 s before and 3 min after adding TG. Samples were subjected to SDS-PAGE and Western
blotting with the specific phospho-p44/42 ERK monoclonal antibody (E10)
as described under "Experimental Procedures." The panels show
results representative of three independent experiments.
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Fig. 9.
Effects of SNP on restoration of basal
[Ca2+]i in human platelets. A,
fura-2-loaded human platelets were incubated at 37 °C for 1 min in
the presence of 100 µM SNP (SNP) or vehicle
(Control). B, platelets were pretreated for 30 min with 1 µM KT5823 and then incubated for 1 further min
with 100 µM SNP (KT5823 + SNP) or
the vehicle (Control). At the time of experiment, 100 µM EGTA was added. Cells were then stimulated with TG (1 µM) combined with ionomycin (50 nM).
Elevations in [Ca2+]i were determined as
described under "Experimental Procedures." Traces shown are
representative of six independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Grant of Junta de Extremadura-Consejería de
Educación y Juventud and Fondo Social Europeo, Spain.
§ Supported by a National Institutes of Health Minority Access to Research Careers Undergraduate Fellowship and the Minority International Research Training Program.
¶ Supported by fellowship from Ministerio de Educación y Cultura/Dirección General de Investigación Científica y Tecnológica, Universidad del Pais Vasco Grants PB94-1357 and UPV 042.310-G11/98, and the Medical Foundation Jesús de Gangoiti-Barrera.
To whom correspondence should be addressed: Dept. of
Physiology, Downing Street, University of Cambridge, Cambridge CB2 3EG, United Kingdom. Tel.: 44-1223-333870; Fax: 44-1223-333840; E-mail: sos10@ cam.ac.uk.
Published, JBC Papers in Press, January 26, 2001 DOI 10.1074/jbc.M009217200
2 J. A. Rosado and S. O. Sage, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SMCE, store-mediated calcium entry; [Ca2+]i, intracellular-free calcium concentration; TG, thapsigargin; PAO, phenylarsine oxide; PBS, phosphate-buffered saline; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; SNP, sodium nitroprusside; PGE1, prostaglandin E1; PMCA, plasma-membrane Ca2+-ATPase; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinases; PAGE, polyacrylamide gel electrophoresis; BAPTA, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid.
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