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J. Biol. Chem., Vol. 275, Issue 26, 19529-19535, June 30, 2000

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Regulation of Plasma Membrane Ca²⁺-ATPase by Small GTPases and Phosphoinositides in Human Platelets^*

Juan A. Rosado and Stewart O. Sage^§

From the Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom

Received for publication, February 16, 2000, and in revised form, March 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have investigated the restoration of [Ca²⁺]_i in human platelets following the discharge of the intracellular Ca²⁺ stores. We found that the plasma membrane Ca²⁺-ATPase is the main mechanism involved in Ca²⁺ extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S-geranylgeranyl-L-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca²⁺]_i to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca²⁺ extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca²⁺]_i. Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca²⁺-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca²⁺ extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca²⁺-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca²⁺]_i to basal levels, suggesting that the products of these kinases are involved in stimulating Ca²⁺ extrusion in human platelets.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cytosolic Ca²⁺ concentration ([Ca²⁺]_i)¹ controls a large number of cellular processes ranging from short term responses such as contraction and secretion to longer term modulation of cell growth (1). [Ca²⁺]_i is increased by the release of Ca²⁺ from intracellular stores or by Ca²⁺ entry through plasma membrane Ca²⁺ channels. Removal of Ca²⁺ from the cytosol and the maintenance of low resting [Ca²⁺]_i is mainly mediated by two mechanisms, sequestration of Ca²⁺ into intracellular compartments and Ca²⁺ extrusion across the plasma membrane. Ca²⁺ efflux occurs by two pathways: Na⁺/Ca²⁺ exchange and active transport via the plasma membrane Ca²⁺-ATPase (PMCA) (e.g. Ref. 1).

As in red blood cells and pancreatic acinar cells, the PMCA is the main mechanism for Ca²⁺ extrusion in human platelets at resting Ca²⁺ concentrations (2-4). Hence the PMCA is a key point for the regulation of Ca²⁺ homeostasis in these cells. The activity of PMCA has been shown to be regulated by several mechanisms including Ca²⁺/calmodulin, acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), protein kinases A and C, and by proteases, whose effects have been described as "the last line of defense against sustained high levels of Ca²⁺" (1, 5). Recently it has been shown that tyrosine phosphorylation of platelet PMCA leads to a substantial inhibition of its Ca²⁺-ATPase activity (6). This mechanism, together with the reported role of tyrosine kinases in the activation of store-operated Ca²⁺ entry in different cell types (7, 8) including platelets (9, 10), may serve as positive feedback to increase intracellular calcium concentration during platelet activation.

Small GTPases of the Ras superfamily play a pivotal role in several signal transduction pathways (11). Platelets contain several members of the Ras superfamily of small GTPases as well as upstream regulators and downstream effectors. Many of these molecules are phosphorylated on tyrosine residues during platelet activation, indicating a close relationship between Ras proteins and tyrosine kinases (11). Ras proteins have been reported to play an important role in Ca²⁺ metabolism. These proteins have been shown to regulate the activation of store-mediated Ca²⁺ entry in platelets (12) and other cells (13, 14). In human platelets, this mechanism is partially mediated by the reorganization of the actin cytoskeleton, so providing a physical but reversible interaction between the Ca²⁺ compartments and the plasma membrane (15).

In the present work we have investigated the possibility that small GTPases of the Ras superfamily are involved in the regulation of PMCA activity in human platelets, so providing a regulatory pathway for a more rapid and sustained increase in intracellular calcium during platelet activation. The effects of LY294002 and inhibitors of actin polymerization were also examined to assess the influence of phosphoinositide kinases and the actin cytoskeleton, which have been reported to mediate some intracellular responses downstream of Ras proteins (11, 12), in the activity of PMCA in these cells.

	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, lanthanum chloride, N-methyl-D-glucamine, and thapsigargin (TG) were from Sigma (Poole, Dorset, United Kingdom). Ionomycin (IONO), cytochalasin D (Cyt D), LY294002, oligomycin, antimycin A, and C3 exoenzyme were from Calbiochem (Nottingham, UK). Farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) were from Alexis (Nottingham, United Kingdom). Latrunculin A (Lat A) was from Biomol (Plymouth Meeting, PA). Protein A-agarose was from Upstate Biotechnology (Lake Placid, NY). Anti-phosphotyrosine monoclonal antibody (PY20) was from Transduction Laboratories (Lexington, KY). Anti-PMCA monoclonal antibody (5F10) was from Affinity Bioreagents (Neshanic Station, NJ). Horseradish peroxidase-conjugated ovine anti-mouse IgG antibody (NA931) was from Amersham Pharmacia Biotech. All other reagents were of analytical grade.

Platelet Preparation-- Fura-2-loaded platelets were prepared as described previously (15). Briefly, blood was obtained from healthy drug-free 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. Cells were then collected by centrifugation at 350 × g for 20 min and resuspended in HEPES-buffered saline containing (HBS 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. In some experiments 145 mM N-methyl-D-glucamine hydrochloride replaced NaCl.

Measurement of [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. (16). To compare the rate of decay of [Ca²⁺]_i to basal values after platelet stimulation between different treatments we used the constant of the exponential decay. Traces were fitted to the equation y = A(1 e^K₁T)e^K₂T, where K₂ is the constant of the exponential decay.

Measurement of F-actin Content-- F-actin content of resting and activated platelets was determined according to the modifications (17) of a previously published procedure (18). 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. Finally, platelets were 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 90 s at 3000 × g and resuspended in PBS. Staining of 2 × 10⁷ cells/ml was measured using a Perkin-Elmer Fluorescence Spectrophotometer (Perkin-Elmer, Norwalk, CT). Samples were excited at 496 nm and emission was at 516 nm.

Immunoprecipitation-- Washed platelets (2.5 × 10⁹ cells/ml) were incubated with TG (1 µM) plus IONO (50 nM) in HEPES-buffered saline for the times indicated. Platelets were then lysed with an equal volume of 2 × lysis buffer (1% (w/v) Triton X-100, 158 mM NACL, 10 MM TRIS, 1 MM EGTA, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate containing 1 mM Na_3VO4, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 5 mM benzamidine, pH 7.2). Insoluble cell debris was removed by centrifugation at 4 °C for 5 min at 16,000 × g. Cell lysates were precleared by incubation with Protein A-agarose for 1 h. After removal of the Protein A-agarose by centrifugation at 16,000 × g for 1 min, 4 µg of anti-phosphotyrosine monoclonal antibody (PY20) was added followed by 2 h incubation. Protein A-agarose (50 µl) was then added and incubation continued for an additional 1 h. The immunoprecipitates were then collected by centrifugation at 16,000 × g for 1 min and washed three times with wash buffer (1% (w/v) Triton X-100, 158 mM NaCl, 10 mM Tris, 1 mM EGTA, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate containing 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, pH 7.2). Finally the pellet was resuspended in Laemmli's buffer (19) and analyzed further by SDS-polyacrylamide gel electrophoresis and Western blotting.

Western Blotting-- Proteins were electrophoresed on 7.5% SDS-polyacrylamide gels and electrophoretically transferred, for 2 h at 0.8 mA/cm², in a semi-dry blotter (Hoefer Scientific, Newcastle Staffs., UK) onto nitrocellulose membranes 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. Membranes were then incubated for 1 h with anti-PMCA monoclonal antibody (5F10) diluted 1:500 in TBST. 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. The density of bands on the film was measured using a Quantimet 500 densitometer (Leica, Milton Keynes, UK).

Statistical Analysis-- Analysis of statistical significance was performed using Student's unpaired t test. For multiple comparison, one-way analysis of variance combined with the Dunnett tests was used.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ Extrusion in Human Platelets-- Fig. 1A represents schematically the main Ca²⁺ transport systems involved in Ca²⁺ signaling during platelet activation. The sequential activation of these systems results in changes in [Ca²⁺]_i. Using certain experimental conditions similar to those previously applied to pancreatic acinar cells by Toescu and Petersen (20) we have ascertained the importance of Ca²⁺ extrusion during Ca²⁺ signaling in human platelets. In a Ca²⁺-free medium (100 µM EGTA added), inhibition of the endomembrane Ca²⁺-ATPase (sarco/endoplasmic reticulum Ca²⁺ ATPase) using 1 µM TG in the presence of a low concentration of IONO (50 nM; required for extensive depletion of the intracellular Ca²⁺ stores in platelets where two Ca²⁺ stores with high and low Ca²⁺ leakage rates have been described (21, 22)), resulted in a transient increase in [Ca²⁺]_i due to release of Ca²⁺ from intracellular stores (Fig. 1B, trace a). It has been shown that lanthanum effectively seals the cell at a concentration of 1 mM, blocking both Ca²⁺ entry and extrusion (23). Treatment of platelets with TG plus IONO in the presence of 1 mM lanthanum results in a larger and sustained increase in [Ca²⁺]_i than in the absence of lanthanum (Fig. 1B, trace b). As shown in Fig. 1A, Ca²⁺ extrusion is the main mechanism responsible for the difference between the responses observed in the presence (trace b) and the absence of lanthanum (trace a).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Ca2+ extrusion in human platelets. A, schematic presentation of the Ca2+ transport systems involved in human platelet activation. Transport 1, represents the release of Ca2+ from the intracellular stores. Transport 2, indicates the uptake of Ca2+ into the Ca2+ stores. Transport 3, shows Ca2+ influx from the extracellular medium. Transport 4, represents Ca2+ extrusion across the plasma membrane. Information about Ca2+ extrusion can be obtained from the difference between protocols a and b. B, Fura-2-loaded human platelets were stimulated with TG (1 µM) plus IONO (50 nM) in a Ca2+-free medium in the absence (a) or presence (b) of 1 mM LaCl3. Elevations in [Ca2+]i were monitored using the 340/380 fluorescence ratio and traces were calibrated in terms of [Ca2+]i. Traces are representative of five independent experiments.

To confirm extrusion can occur against a concentration gradient and to check whether the decrease in [Ca²⁺]_i could be partially mediated by Ca²⁺ leakage from the cells we performed a series of experiments in the presence of 500 nM extracellular Ca²⁺. Fig. 2A shows the effects of treating platelets with 1 µM TG plus 50 nM IONO in a Ca²⁺-free medium (100 µM EGTA added) or when 500 nM Ca²⁺ was added. Although [Ca²⁺]_i was slightly higher after stimulation in the presence of external Ca²⁺, an effect that is mediated by Ca²⁺ entry, the pattern of decay under both conditions was similar (the rate of decay was 0.0078 ± 0.0008 in a Ca²⁺-free medium and 0.0076 ± 0.0006 in a medium containing 500 nM Ca²⁺) and the [Ca²⁺]_i 5 min after the addition of TG plus IONO was also comparable (Fig. 2A; n = 5).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of low [Ca2+]o, Na+ replacement or mitochondrial inhibitors on restoration of [Ca2+]i in human platelets. A, Fura-2-loaded human platelets were stimulated with TG (1 µM) combined with IONO (50 nM). At the time of experiment, 100 µM EGTA or 500 nM CaCl2 was added. At the end of the experiment, the cells in a medium containing 500 nM CaCl2 were lysed with 0.01% (v/v) Triton X-100 to give an indication of extracellular Ca2+ concentration. B, cells were stimulated with TG plus IONO in normal HBS (Control) or in a medium in which Na+ was completely replaced by N-methyl-D-glucamine (Na+-free). C, platelets were stimulated with TG plus IONO in normal HBS (Control) or in HBS supplemented with 10 mM antimycin A and 10 µg/ml oligomycin (A+O). Elevations in [Ca2+]i were determined as described in the legend to Fig. 1. Traces shown are representative of five separate experiments.

Established routes for Ca²⁺ extrusion are Na⁺/Ca²⁺ exchange and the PMCA. Although the main mechanism for Ca²⁺ extrusion at low Ca²⁺ concentration has been reported to be the PMCA (2, 20) we also studied the possible involvement of the Na⁺/Ca²⁺ exchanger in the removal of Ca²⁺ from the cytosol. Fig. 2B shows the response evoked by 1 µM TG plus 50 nM IONO in control medium (HBS) and when the cells were suspended in medium in which all Na⁺ had been replaced by N-methyl-D-glucamine. In both cases, [Ca²⁺]_i was elevated to the same levels and then fell back to basal. The rate of decay of [Ca²⁺]_i to basal levels was 0.0094 ± 0.0002 in N-methyl-D-glucamine buffer and 0.0092 ± 0.0002 in paired controls (n = 5).

Mitochondria have been reported to remove Ca²⁺ from the cytosol, modulating physiological and pathophysiological cytosolic responses (24). Hence we studied whether Ca²⁺ uptake by mitochondria could be involved in removal of Ca²⁺ from the platelet cytosol under our experimental conditions. Fig. 2C shows the responses evoked by 1 µM TG plus 50 nM IONO in control medium (HBS) and when platelets were suspended in HBS supplemented with 10 µM antimycin A and 10 µg/ml oligomycin to eliminate Ca²⁺ uptake by mitochondria (25). As shown in Fig. 2C, the presence of antimycin A and oligomycin did not alter the pattern of decay in the [Ca²⁺]_i to basal levels. The rate of decay was 0.0098 ± 0.0003 in normal HBS and 0.0096 ± 0.0001 in HBS supplemented with antimycin A and oligomycin (n = 5). Taken together, these results indicate that under our experimental conditions the PMCA is the main mechanism involved in Ca²⁺ removal from the cytosol by extrusion across the plasma membrane.

Role of Small GTPases in PMCA Activity-- We have recently reported that farnesylcysteine analogs impair membrane association of the small GTPases of the Ras superfamily by preventing methylation of farnesylated or geranylgeranylated proteins (12), a process that is required for the activation of these GTPases (26). In the present study we used FTA, an agent that selectively prevents methylation of farnesylated Ras proteins like Ras, Rac, Rap 1a, and Rap 2a, and AGGC, which inhibits methylation of geranylgeranylated proteins, such as Rab, Rho, Rap1b, or Rap 2b (27), to investigate the possible role of Ras proteins in PMCA activation. Fig. 3A shows the effect of preincubation at 37 °C for 20 min with 40 µM FTA combined with 30 µM AGGC on the response evoked by TG (1 µM) plus IONO (50 nM). Preincubation with FTA plus AGGC did not modify the release of Ca²⁺ from the intracellular stores stimulated by TG plus IONO; however, the rate of decay was enhanced about 2-fold. The decay constants were 0.0156 ± 0.0018 in FTA plus AGGC-treated platelets and 0.0070 ± 0.0001 in paired controls (n = 6; p < 0.01).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of Ras protein inhibitors on restoration of [Ca2+]i in human platelets. A, Fura-2-loaded human platelets were incubated at 37 °C for 20 min in the presence of 40 µM FTA combined with 30 µM AGGC (FTA+AGGC) or the vehicle (Control). B, platelets were incubated at 37 °C for 2 h with 100 µg/ml C. botulinum C3 exoenzyme (C3 exoenzyme) or the vehicle (Control). At the time of experiment, 100 µM EGTA was added. Cells were then stimulated with TG (1 µM) combined with IONO (50 nM). Elevations in [Ca2+]i were determined as described in the legend to Fig. 1. Traces shown are representative of four to six independent experiments.

To further investigate the role of small GTPases in PMCA activity we examined the effect of Clostridium botulinum C3 exoenzyme. C3 exoenzyme is a 25-kDa enzyme that has been shown to inactivate Rho A by ADP-ribosylation (28). As shown in Fig. 3B, treatment of human platelets for 2 h at 37 °C with 100 µg/ml C. botulinum C3 exoenzyme did not alter either TG plus IONO-induced Ca²⁺ release from the intracellular stores or the rate of decay of [Ca²⁺]_i to basal levels. The decay constants are 0.0070 ± 0.0001 and 0.0067 ± 0.0002 for control or C3 exoenzyme-treated platelets, respectively (n = 4). We have previously shown that treatment of platelets with C3 exoenzyme is effective at inhibiting store-regulated Ca²⁺ entry (12).

Several members of the Ras superfamily of small GTPases are believed to regulate the organization of the actin cytoskeleton. Treatment of platelets for 20 min with 40 µM FTA combined with 30 µM AGGC significantly reduced actin filament formation induced by TG (1 µM) plus IONO (50 nM) (Table I). To investigate whether the actin cytoskeleton plays a role in Ca²⁺ extrusion we investigated the effect of Cyt D and Lat A, two agents that inhibit actin filament polymerization by different mechanisms (29, 30). Human platelets were pretreated with 10 µM Cyt D for 40 min at 37 °C and actin filament content was determined using fluorescein isothiocyanate-phalloidin. As shown in Table I, Cyt D (10 µM) was without effect on the actin filament content of unstimulated platelets, but prevented TG plus IONO-induced actin filament formation. As shown in Fig. 4A, treatment of platelets with 10 µM Cyt D for 40 min did not modify the rate of decay of the [Ca²⁺]_i to basal levels. The decay constants were 0.0059 ± 0.0007 and 0.0061 ± 0.0005 for control and Cyt D-treated platelets, respectively (n = 5). Similar results were obtained using Lat A. Preincubation of platelets for 1 h at 37 °C with 3 µM Lat A abolished TG plus IONO-induced actin polymerization without modifying actin filament content in non-stimulated platelets (Table I). As for Cyt D, Lat A did not significantly modify the rate of decay of the [Ca²⁺]_i to basal levels. The decay constants were 0.0051 ± 0.0004 in Lat A-treated platelets and 0.0045 ± 0.0002 in paired controls (n = 5; p = 0.36).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of cytochalasin D and latrunculin A on restoration of [Ca2+]i in human platelets. A, Fura-2-loaded human platelets were incubated at 37 °C for 40 min in the presence of 10 µM cytochalasin D (Cyt D) or the vehicle (Control). B, platelets were incubated at 37 °C for 1 h with 3 µM latrunculin A (Lat A) or the vehicle (Control). At the time of experiment, 100 µM EGTA was added. Cells were then stimulated with TG (1 µM) combined with IONO (50 nM). Elevations in [Ca2+]i were determined as described in the legend to Fig. 1. Traces shown are representative of five independent experiments.

Effect of Small GTPases on Tyrosine Phosphorylation of PMCA in Human Platelets-- Tyrosine phosphorylation of PMCA has recently been reported to inhibit its activity (6). Activation of human platelets with 1 µM TG combined with 50 nM IONO caused a rapid tyrosine phosphorylation of PMCA (Fig. 5A). An increase in tyrosine phosphorylation was detected 1 min after addition of TG plus IONO, reached a maximum within 3 min with a 2.46 ± 0.2-fold increase, and was maintained for at least 10 min (Fig. 5A; n = 4).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effects of FTA plus AGGC on tyrosine phosphorylation of PMCA in human platelets. Platelets (2.5 × 109 cells/ml) were stimulated with TG (1 µM) combined with IONO (50 nM) and aliquots were removed at the indicated times and lysed. Whole cell lysates were immunoprecipitated with anti-phosphotyrosine monoclonal antibody (PY20). Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by transfer of proteins onto a nitrocellulose membrane and anti-PMCA immunoblotting as described under "Experimental Procedures." Bands were revealed using chemiluminescence, and quantification of phosphorylation was performed by scanning densitometry. A, time course of tyrosine phosphorylation of PMCA in TG plus IONO-stimulated platelets. The top panel shows results from a representative experiment. The arrow represents the position of PMCA. In the lower panel data shown are presented as mean ± S.E. of four independent experiments and are expressed as -fold increase over the pretreatment level (experimental/control). B, platelets were pretreated for 20 min at 37 °C in the absence or presence of 40 µM FTA combined with 30 µM AGGC. Cells were then stimulated for a further 3 min with no addition or with TG plus IONO. The top panel shows results from one experiment representative of three others. The arrow represents the position of PMCA. In the lower panel data are presented as mean ± S.E. and are expressed as percentages of control (untreated platelets).

We have recently reported that farnesylcysteine analogs do not have a general inhibitory effect on tyrosine kinase activity in human platelets (12). However, pretreatment of human platelets with 40 µM FTA plus 30 µM AGGC for 20 min at 37 °C significantly decreased the tyrosine phosphorylation of the PMCA stimulated by treatment with TG plus IONO by 85.6 ± 3.67% (Fig. 5B; p < 0.001; n = 4). Pretreatment of platelets for 20 min with FTA plus AGGC did not significantly modify basal tyrosine phosphorylation of PMCA (Fig. 5B).

Role of Phosphoinositide Kinases on PMCA Activation-- Phosphoinositide 3-kinase (PI 3-kinase) and phosphoinositide 4-kinase (PI 4-kinase) have been shown to be regulated upstream by small GTPases in platelets and other cells (11, 31). To investigate the involvement of these kinases in Ca²⁺ extrusion we examined the effect of LY294002, a cell permeant specific inhibitor of PI 3-kinase and PI 4-kinase (32, 33). We have previously demonstrated that treatment of human platelets for 30 min with LY294002 inhibits PI 3-kinase and PI 4-kinase activity in a concentration-dependent manner reaching a complete inhibition of PI 3-kinase activity at a concentration of 10 µM and almost complete inhibition of PI 4-kinase activity at a concentration of 100 µM (33). Treatment of platelets for 30 min at 37 °C with 10 or 100 µM LY294002 did not modify Ca²⁺ release from the intracellular stores induced by TG plus IONO. However, LY294002 significantly decreased the rate of decay of [Ca²⁺]_i to basal levels at both concentrations investigated. The decay constant was reduced from 0.0070 ± 0.0003 in control platelets to 0.0052 ± 0.0005 or 0.0048 ± 0.0002 when platelets were preincubated with 10 or 100 µM LY294002, respectively (Fig. 6; p < 0.01; n = 5).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of LY294002 on restoration of [Ca2+]i in human platelets. Fura-2-loaded human platelets were incubated for 30 min at 37 °C in the presence of 10 or 100 µM LY294002 or the vehicle (Control). At the time of experiment, 100 µM EGTA was added. Cells were then stimulated with TG (1 µM) combined with IONO (50 nM). Elevations in [Ca2+]i were determined as described in the legend to Fig. 1. Traces shown are representative of five separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PMCA plays a key role in [Ca²⁺]_i homeostasis. The PMCA and the Na⁺Ca²⁺ exchanger are the only transporters capable of removing Ca²⁺ from the cell; however, since some studies indicate that the Na⁺/Ca²⁺ exchanger does not significantly contribute to Ca²⁺ efflux in platelets at resting Ca²⁺ levels (2, 34), the active transport via the PMCA might be the only mechanism responsible for the maintenance of low resting [Ca²⁺]_i in these cells.

Our findings show that depletion of intracellular Ca²⁺ stores with the inhibitor of the endomembrane Ca²⁺-ATPase, TG, combined with a low concentration of ionomycin in the presence of 1 mM lanthanum, which has been reported to block both Ca²⁺ entry and extrusion (23), evokes an increase in [Ca²⁺]_i which is sustained and of a higher amplitude than in the absence of lanthanum in a Ca²⁺-free medium. The present data indicate that Ca²⁺ extrusion, which was found to be very powerful, is rapidly activated following the rise in [Ca²⁺]_i after depletion of the intracellular stores. Ca²⁺ extrusion can occur against a concentration gradient as demonstrated by repetition of our basic observations under conditions where [Ca²⁺]_o was set at 500 nM.

In agreement with previous reports (2, 34), our results indicate that Na⁺/Ca²⁺ exchange has a negligible, if any effect in the restoration of [Ca²⁺]_i to low resting levels when a rise in [Ca²⁺]_i was induced by depletion of the intracellular Ca²⁺ stores using TG and a low concentration of IONO. Therefore, as in a number of cell types (2, 20, 34), under our experimental conditions the main Ca²⁺ extrusion system in human platelets is the PMCA. In addition, we found that Ca²⁺ uptake by mitochondria is negligible under our experimental conditions. These results are in agreement with previous studies in platelets and other cells (35, 36), which suggest that mitochondria might have a greater role at a higher cytosolic Ca²⁺ concentration. In summary, our findings, in agreement with previous observations, indicate that the removal of Ca²⁺ from the platelet cytosol is exclusively mediated by the activity of the PMCA under our conditions where the sarco/endoplasmic reticulum Ca²⁺-ATPase is inhibited by TG (2).

Human platelets have been reported to express both the PMCA1 and PMCA4 isoforms (6). PMCA is a multiregulated transporter. The activity of PMCA can be stimulated by protein kinases, such as protein kinases C and A, proteases like calpain, which has been suggested to be an in vivo regulator of [Ca²⁺]_i, calmodulin, and acidic phospholipids like PtdIns(4,5)P₂ (1, 5). On the other hand, tyrosine kinases have been reported to inhibit the PMCA, which shows a substantial basal activity (6). We now provide evidence for the involvement of small GTPases of the Ras superfamily in the regulation of PMCA activity.

The Ras superfamily of small GTPases have been shown to be prenylated (farnesylated or geranylgeranylated) and methylated, an essential process for the association of Ras protein with membranes, which plays an important role in their activation (26). We have recently reported that farnesylcysteine analogs, specific inhibitors of the methylation of small G-proteins, impaired the membrane association of Ras proteins in human platelets (12). The results presented here demonstrate that treatment of platelets with FTA combined with AGGC, specific inhibitors of the methylation of farnesylated and geranylgeranylated Ras proteins, respectively (38), significantly increases the rate of decay of [Ca²⁺]_i to resting levels, a phenomenon that could be explained by an increase in Ca²⁺ extrusion by the PMCA. These findings indicate that PMCA activity is negatively regulated by the small GTPases of the Ras superfamily.

It remains to elucidate how small GTPases might regulate the activity of PMCA. Several cell functions are regulated by Ras proteins through the reorganization of the actin cytoskeleton (39). In fact, we have found that treatment of platelets with FTA combined with AGGC abolished TG plus IONO-induced actin polymerization. To determine whether the actin cytoskeleton is important in mediating the activation of PMCA in human platelets we used two inhibitors of actin polymerization, Cyt D, which inhibits actin polymerization by preventing monomer addition at the growing end of the polymer (40), and Lat A, which inhibits actin polymerization by a different mechanism. The effects of Cyt D and Lat A on the cytoskeleton were confirmed by measurement of the actin filament content of platelets. Treatment of platelets with Cyt D or Lat A completely inhibited actin polymerization induced by TG plus IONO without having any significant effect on the actin filament content of resting platelets. Platelets treated with Cyt D or Lat A retained their ability to respond to TG and ionomycin and the restoration of [Ca²⁺]_i to resting levels was not found to be different from that in non-treated cells, suggesting that the actin cytoskeleton is not a modulator of the PMCA activity. Our results are in agreement with the observations of Dean et al. (6), who reported that the PMCA is not associated with the cytoskeleton in human platelets.

Consistent with these observations, inactivation of Rho A, a small GTPase of the Ras family that regulates the organization of the actin filament network (41), using C. botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation (28) demonstrates that Rho A is not involved in the regulation of PMCA activity. Treatment of platelets with C3 exoenzyme did not alter either the release of Ca²⁺ from the intracellular stores or the rate of restoration of the [Ca²⁺]_i to resting levels.

It has been reported that tyrosine phosphorylation of PMCA in platelets leads to inhibition of its Ca²⁺-ATPase activity below the basal level (6). Treatment of platelets with TG and IONO increased tyrosine phosphorylation of PMCA in a time-dependent manner, reaching a maximum after 3 min of stimulation that was maintained for up to 10 min. These findings show similarities to and differences from the observations of Dean et al. (6), who showed that thrombin increased tyrosine phosphorylation of PMCA with a maximum effect at 5 min; however, after 10 min of stimulation the level of phosphorylation had returned nearly to basal. This difference can be explained by the lack of refilling of the intracellular stores under our experimental conditions, since TG is present in the medium throughout the experiment. It has been suggested that store refilling activates a tyrosine phosphatase (42). This could mediate the reduction of tyrosine phosphorylation of PMCA 5 min after stimulation with thrombin. Since tyrosine phosphorylation of PMCA is maintained even when the [Ca²⁺]_i is returned to basal levels, our results suggest that tyrosine phosphorylation of PMCA is not dependent on sustained increases in [Ca²⁺]_i, although our experimental conditions do not allow us to estimate the level of Ca²⁺ in the proximity of the pump, that has been shown to be located in specific areas, caveolae, where changes in free Ca²⁺ may be even greater (e.g. Ref. 1). Furthermore, we cannot exclude the possibility that Ca²⁺ elevation serves as an initiator for this process. To investigate whether the inhibitory role of Ras proteins on PMCA activity could be mediated by tyrosine phosphorylation of the pump, we examined the effect of FTA combined with AGGC. Our results show that treatment with FTA plus AGGC markedly inhibited the tyrosine phosphorylation of PMCA induced by TG and IONO. This observation indicates that Ras proteins are involved in negative regulation of PMCA activity and that the mechanism may involve tyrosine phosphorylation of the pump.

Acidic lipids, such as PtdIns(4,5)P₂, have been reported to be potent activators of the erythrocyte PMCA; however, other derivatives of the PtdIns cycle-like diacylglycerol showed a negligible effect (43). PtdIns(4,5)P₂ is important in keeping the PMCA partially active in resting cells (44). We have recently reported that the products of PI 3-kinase and PI 4-kinase (PtdIns(3)P, PtdIns(4)P, or their derivatives) are involved in Ca²⁺ responses through the regulation of Ca²⁺ entry in human platelets (33). In addition, PI 4-P is the immediate precursor of PI(4,5)P₂, which is required for the agonist-evoked release of Ca²⁺ from intracellular stores (e.g. 37). Hence, we have evaluated the role of PI 3- and PI 4-kinases in the regulation of PMCA activity using the inhibitor LY294002. We have previously reported that treatment of human platelets for 30 min with LY294002 effectively abolished PI 3-kinase activity at a concentration 10 µM. In addition, this agent reduced PI 4-kinase activity by 80% at a concentration 100 µM (33). Treatment of platelets with LY294002 also resulted in a concentration-dependent reduction in the level of PtdIns phosphate with an IC₅₀ of 22.7 µM (33). The present data show that LY294002 reduces Ca²⁺ removal from the platelet cytosol at concentrations that inhibit the activity of both PI 3- and PI 4-kinases. The effect of LY294002 was found to be concentration-dependent with a larger reduction in the rate of decay of [Ca²⁺]_i to resting levels at 100 µM and a smaller but significant effect at 10 µM. The effect of the highest concentration of LY294002 (100 µM) could be explained by an effect of PtdIns(4)P itself on the activity of PMCA or, on the basis of its role as a precursor for PtdIns(4,5)P₂, its effect could be mediated by a decrease in the level of PtdIns(4,5)P₂. However, since PtdIns(3)P is not a precursor of PtdIns(4,5)P₂, the effect observed using LY294002 at a concentration that specifically inhibits PI 3-kinase activity (10 µM LY294002 had a negligible effect on PI 4-kinase activity (33)) is likely to be explained by a role for the products of this kinase on PMCA activity.

These observations presented in this report indicate the importance of small GTPases of the Ras superfamily in facilitating elevations in the [Ca²⁺]_i after stimulation. We have previously reported that Ras proteins are required both for the activation and maintenance of Ca²⁺ influx in human platelets (12). Our new data indicate that these small GTPases also exert a negative regulation on PMCA activity. Taken together, these results suggest that the activation of Ras proteins might serve as a signal leading to the sustained increases in [Ca²⁺]_i required for refilling of intracellular Ca²⁺ pools and the activation of Ca²⁺-dependent pathways. Our earlier work indicated that the activation of PI 3- and PI 4-kinases is also involved in the activation but not the maintenance of Ca²⁺ entry in platelets (33). The present results indicate that these lipid kinases are also involved in the activation of PMCA. This suggests a role for PI 3- and PI 4-kinases in mediating rapid but transient elevations, rather than sustained increases in [Ca²⁺]_i in human platelets.

	FOOTNOTES

^* This work was supported in part by The Wellcome Trust Grant 051560.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.

^§ 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, March 31, 2000, DOI 10.1074/jbc.M001319200

	ABBREVIATIONS

The abbreviations used are: [Ca²⁺]_i, intracellular free calcium concentration; TG, thapsigargin; IONO, ionomycin; PBS, phosphate-buffered saline; HBS, HEPES-buffered saline; Cyt D, cytochalasin D; Lat A, latrunculin A; PtdIns, phosphatidylinositol; FTA, farnesylthioacetic acid; AGGC, N-acetyl-S-geranylgeranyl-L-cysteine; PMCA, plasma membrane Ca²⁺ ATPase; [Ca²⁺]_o, extracellular calcium concentration; PI 3-kinase, phosphatidylinositol 3-kinase; PI 4-kinase, phosphatidylinositol 4-kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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