Cyclic Nucleotides Modulate Store-mediated Calcium Entry through the Activation of Protein-tyrosine Phosphatases and Altered Actin Polymerization in Human Platelets*

Juan A. RosadoDagger, Tanya Porras§, Manuel Conde, and Stewart O. Sage||

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/PKBalpha (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.

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/PKBalpha Ser473 Phosphorylation-- Phosphorylation of Akt/PKBalpha on the residue Ser473 was assessed by SDS-PAGE and Western blotting as described above but using a specific anti-phospho-Akt/PKBalpha (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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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.

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).


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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.

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).


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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.

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.


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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.

                              
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Table I
Effects of PGE1 or SNP on the F-actin content of platelets in which store-mediated calcium entry had been preactivated
Dimethyl-BAPTA-loaded human platelets were suspended in a Ca2+-free medium 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 were added. Samples were removed 5 s before adding TG and 1 min after the addition of PGE1, SNP, or the vehicle and F-actin content was determined as described under "Experimental Procedures." Values given are the F-actin content expressed as a percentage of the basal and are presented as mean ± S.E. of six separate determinations.

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).


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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.

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.

                              
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Table II
Effects of PKA, PKG, and protein-tyrosine phosphatases on actin polymerization in platelets
Dimethyl-BAPTA-loaded human platelets were preincubated for 30 min at 37 °C with 3 µM KT5720, 1 µM KT5823, 100 µM sodium vanadate, 10 µM PAO or the vehicles. Cells were then incubated for 1 min with 5 µM PGE1, 100 µM SNP, or the vehicle and stimulated with either TG (200 nM), thrombin (0.1 unit/ml) or ADP (40 µM). Samples were removed 5 s before and 3 min after the addition of the agonists and F-actin content was determined as described under "Experimental Procedures." Values given are the changes in F-actin content expressed as a percentage of the increases in controls (agonist-induced actin polymerization in the absence of treatment) and are presented as mean ± S.E. of six separate determinations.

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).


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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.

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).


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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.

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.

                              
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Table III
Effects of PKA, PKG, or protein-tyrosine phosphatases on TG-stimulated store-mediated Ca2+ entry
Fura-2-loaded human platelets were incubated for 30 min at 37 °C with 3 µM KT5720, 1 µM KT5823, 100 µM sodium vanadate, 10 µM PAO or the vehicles. At the time of the experiment 100 µM EGTA was added. Cells were then incubated for 1 min with 5 µM PGE1, 100 µM SNP or the vehicle and 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." Data indicate the percentage of Ca2+ entry relative to respective controls (vehicle was added). Ca2+ entry was estimated as described under "Experimental Procedures." Values are mean ± S.E. of six to ten separate determinations.

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).

                              
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Table IV
Effects of PKA, PKG, or protein-tyrosine phosphatases on thrombin-stimulated Ca2+ elevations in the presence or absence of external Ca2+
Fura-2-loaded human platelets were incubated for 30 min at 37 °C with 3 µM KT5720, 1 µM KT5823, 100 µM sodium vanadate, 10 µM PAO, a combination of KT5720 or KT5823 plus sodium vanadate or PAO or the vehicles. At the time of the experiment 1 mM Ca2+ or 100 µM EGTA was added. Cells were then incubated for 1 min with 5 µM PGE1 or 100 µM SNP and stimulated with thrombin (0.1 unit/ml). Elevations in [Ca2+]i were monitored using the 340/380 nm ratio and thrombin-induced Ca2+ elevations and release were estimated by integration as described under "Experimental Procedures." Data indicate the percentage of Ca2+ elevation or release relative to controls (vehicle was added). Values are mean ± S.E. of six determinations.

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/PKBalpha on Ser473. It is well known that activation of PI3K results in phosphorylation of Akt/PKBalpha 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/PKBalpha 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/PKBalpha 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/PKBalpha 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.

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).


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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

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).

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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