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(Circulation Research. 1999;84:201-209.)
© 1999 American Heart Association, Inc.

Original Contribution

Nitric Oxide Inhibits Capacitative Cation Influx in Human Platelets by Promoting Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPase–Dependent Refilling of Ca²⁺ Stores

Elena S. Trepakova, Richard A. Cohen, Victoria M. Bolotina

From the Vascular Biology Unit, Department of Medicine, Boston University Medical Center, Boston, Mass.

Correspondence to Dr Victoria M. Bolotina, Vascular Biology Unit, R-408, Boston University Medical Center, 88 E Newton St, Boston, MA 02118. E-mail vbolotina{at}med-med1.bu.edu

	Abstract

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Abstract—Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca²⁺ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca²⁺/Mn²⁺ influx in intact platelets and to monitor Ca²⁺ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca²⁺ and Mn²⁺ influx developed rapidly (within 30 s) after fast release of Ca²⁺ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca²⁺ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) with 30 µmol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 µmol/L) inhibited capacitative Ca²⁺ and Mn²⁺ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca²⁺ and Mn²⁺ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca²⁺-free medium, NO reduced the peak Ca²⁺ rise caused by thrombin and significantly promoted Ca²⁺ back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 µmol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca²⁺ concentration. Inhibition of Ca²⁺ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca²⁺ into the stores after inositol-1,4,5-trisphosphate–induced Ca²⁺ release or after addition of Ca²⁺ (1 µmol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca²⁺ uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca²⁺ stores.

Key Words: nitric oxide • sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase • cation influx • Ca²⁺ • platelets

	Introduction

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An elevation in cytoplasmic free Ca²⁺ concentration (Ca²⁺_cyt) is a major component of the signal transduction following receptor stimulation by thrombin in platelets.¹ Thrombin activates phospholipase C, which generates inositol-1,4,5-trisphosphate (IP₃) and leads to the rapid depletion of IP₃-sensitive Ca²⁺ stores^{2 3} and initiation of cation influx.^{4 5 6} Similar to other types of nonexcitable cells,⁷ agonist-activated Ca²⁺ influx in platelets is thought to be capacitative in nature, being also activated by passive store depletion with an inhibitor(s) of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), thapsigargin (TG) or 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ).^{8 9 10 11}

Nitric oxide (NO), a potent inhibitor of thrombin-activated platelet aggregation,^{12 13} is known to decrease Ca²⁺_cyt that is raised by agonists,¹⁴ although the mechanism is not well defined. It has been shown that NO inhibits thrombin- and IP₃-stimulated Ca²⁺ release from internal stores in intact¹⁵ and permeabilized platelets.^{16 17} However, it is still unclear whether NO also inhibits capacitative cation influx. Brune et al¹⁷ found a significant inhibitory effect of the NO donor, sodium nitroprusside, on Mn²⁺ influx in TG-treated platelets. In contrast, Okamoto et al¹⁸ did not find an effect of the NO donor on TG-induced Ca²⁺ influx in human platelets and concluded that capacitative Ca²⁺ influx is resistant to NO.

The data presented here demonstrate that NO, indeed, is a potent inhibitor of capacitative cation influx in human platelets and that SERCA activity is required for this effect. This finding is consistent with the idea that NO inhibits capacitative cation influx indirectly by promoting SERCA-dependent refilling of intracellular Ca²⁺ stores.

	Materials and Methods

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Platelet Isolation Procedure
Blood from healthy adult volunteers was drawn into plastic nonsterile tubes containing 10% volume of anticoagulant. The composition of anticoagulant was (in g/100 mL): trisodium citrate 2.5, citric acid 1.5, and glucose 2.0. To obtain platelet-rich plasma (PRP), the anticoagulated blood was centrifuged for 10 minutes at 350g at room temperature. Aspirin (100 µmol/L) was added to PRP to inhibit the activation of the platelets by thromboxane A₂. Platelets were used within 5 hours after preparation.

For experiments on permeabilized platelets, cells were obtained by gel filtration as follows. Prewashed Sepharose-2B gel (Sigma) was placed into the plastic column with the 70-µm nylon mesh attached to the bottom and equilibrated with a 3x volume of standard HEPES buffer of the following composition (in mmol/L): NaCl 137, KCl 2.7, MgCl₂ · 6H₂O 1, NaH₂PO₄ 3.3, glucose 5.5, and HEPES 3.8 (pH 7.4). Ca²⁺ was not added to the buffer, but its residual concentration was 1 to 3 µmol/L as measured by Ca²⁺-sensitive electrode. Buffer was supplemented with 100 µmol/L aspirin and 6 mU/mL apyrase to prevent platelet activation with thromboxane A₂ and traces of ADP, respectively. After collection, the platelet suspension was stored at 37°C and used during the next 3 to 4 hours. The cell number in the suspension used for experiments was 2 to 2.5x10⁸/mL.

Fluorescence Measurements
The fluorescent probe fura-2¹⁹ was used to monitor changes in Ca²⁺_cyt in platelets. PRP was diluted with standard buffer to the final cell number of 10⁸/mL and centrifuged at 750g for 10 minutes. Platelets were resuspended in the same buffer containing 2.5 µmol/L fura-2/AM and incubated at 37°C for 10 minutes. The platelet suspension was then centrifuged at 750g for 10 minutes and resuspended in 2 mL of buffer immediately before the fluorescence measurements. Cells were loaded with fura-2 before each run. Each experiment was done with 6 to 8x10⁷ cells/mL. Fluorescence (F) measurements were carried out at 37°C using a spectrofluorimeter (Hitachi F-4500) with excitation wavelength alternating between 340 and 380 nm every 0.5 s and emission wavelength 510 nm. Changes in Ca²⁺_cyt were estimated from the F₃₄₀/F₃₈₀ ratio (R_340/380). Data collection, ratio calculation, and analysis were performed with the Hitachi software. All the recordings were corrected for the cell autofluorescence determined in unloaded platelets. In some experiments the initial rate of fall in Ca²⁺ concentration caused by NO in thrombin-stimulated platelets, and after addition of IP₃ or Ca²⁺ in permeabilized platelets, was estimated from the initial slope during the first 5 to 20 seconds. Importantly, control experiments showed no evidence for a direct effect of NO either on response of fura-2 to Ca²⁺ or on the fluorescence properties of fura-2. To study cation influx, Mn²⁺ (100 to 300 µmol/L) was added to the platelets suspended in standard buffer, and the rate of fura-2 quenching at an excitation and emission wavelength of 360 and 510 nm, respectively, was determined. The rate of Mn²⁺ influx was estimated from the slope during the first 60 s after Mn²⁺ addition, and the linear fit was performed with Microcal Origin software.

Experiments on Permeabilized Cells
Before the experiment, platelets were resuspended in an artificial cytoplasmic solution containing (in mmol/L): KCl 110, NaCl 10, KH₂PO₄ 1, KH₂CO₃ 5, and HEPES 20 (pH 7.1) and supplemented with 1 µg/mL oligomycin, 2 µg/mL antimycin, 2 mmol/L MgATP, and an ATP-regenerating system containing 5 mmol/L creatine phosphate and 15 U/mL creatine kinase. There was no Ca²⁺ added, and its residual concentration in the cytoplasmic solution was 1 to 3 µmol/L (as measured by a Ca²⁺-sensitive electrode). Fura-2 (K⁺ salt, 1 µmol/L) was added to this solution to monitor the changes in free Ca²⁺. During the experiment 20 µg/mL saponin was applied directly to the recording chamber, causing permeabilization of the platelet plasma membrane and resulting in sequestration of external Ca²⁺ into the stores, lowering the free Ca²⁺ concentration (see Figure 7A). After 4 to 6 minutes, Ca²⁺ concentration stabilized at a level of 200 to 250 nmol/L as calculated from the ratio F₃₄₀/F₃₈₀ assuming a K_d for fura-2 of 224 nmol/L.¹⁹

View larger version (23K): [in this window] [in a new window]   Figure 7. Ca2+ release and uptake in permeabilized platelets. Platelets were suspended in cytosolic solution containing fura-2. A, Changes in free Ca2+ are shown following application of saponin (20 µg/mL), external Ca2+ (1 µmol/L), IP3 (250 µmol/L), and BHQ (10 µmol/L) in the presence of 2 mmol/L ATP. B, The same experiment as in panel A is shown in the presence of heparin (40 µg/mL). C and D, Changes in free Ca2+ in permeabilized platelets following application of IP3 (100 µmol/L) or BHQ (10 µmol/L). NO (1 µmol/L) was applied at the times shown.

NO Solution
A standard 1-L intravenous bag was filled with distilled water (750 mL) that had been bubbled with nitrogen gas to remove oxygen. Approximately 30 mEq of Bio-Rad analytical-grade anion exchange resin was mixed in the water before the bag was filled. The resin retains any nitrites or nitrates that may be formed by the reaction of NO with oxygen. The contents of the bag were bubbled with nitrogen gas for another 30 minutes. The contents were then mixed thoroughly, and the bag was placed in a refrigerator at 4°C. The concentration of NO in the solution equilibrated to give a 3.1±0.6 mmol/L (n=5) saturated solution that was stable at least for 1 week as measured by a chemiluminescent nitric oxide analyzer (Sievers NOA model 270). The absence of contaminating nitrite in saturated NO solution was confirmed by obtaining a similar analysis in the presence and absence of a reducing agent in the reflux chamber of the analyzer (KI in glacial acetic acid). At the time of the experiment, subsequent dilutions were made from the saturated NO solution by drawing off the solution from the bag with a gas-tight syringe and dissolving it in sealed tubes filled with deoxygenated solution (bubbled with nitrogen for 1 hour).

Materials
NO gas was from Matheson. Fura-2/AM and fura-2 (K⁺ salt) were from Molecular Probes. BHQ and IP₃ were from Calbiochem. All other drugs were from Sigma.

Statistical Analysis
Each experiment was repeated 3 to 9 times. ANOVA and paired t test were used to determine the statistical significance of differences in obtained data. P<0.05 was considered significant. The results on the bar graphs are expressed as mean±SE.

	Results

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Cation Influx in Human Platelets Depends on Ca²⁺ Store Emptying
Although the existence of capacitative cation influx in platelets is widely accepted, the only strong evidence for it is the fact that TG and BHQ, inhibitors of SERCA that have been shown to deplete Ca²⁺ stores without elevation of IP₃,²⁰ cause a sustained Ca²⁺ influx^{8 9 10 11} similar to that activated by thrombin.^{4 5 6} To study the effect of NO on capacitative cation influx, the time-dependent relationship between Ca²⁺ store emptying and cation influx was first determined. For this purpose, cation influx after the rapid store emptying caused by thrombin was compared with the influx induced by the slower store emptying caused by inhibition of back-sequestration into the stores by BHQ.

Thrombin-Induced Cation Influx
To experimentally separate intracellular Ca²⁺ release from cation influx from the extracellular space, thrombin was applied at a submaximal concentration (0.5 U/mL) to suspensions of fura-2–loaded platelets in Ca²⁺-free medium. Cation influx was then analyzed following the addition of Ca²⁺ (1 mmol/L) or Mn²⁺ (100 µmol/L). A typical experiment (Figure 1A and 1B) shows that application of thrombin in the absence of extracellular Ca²⁺ caused a very rapid increase in Ca²⁺_cyt that, after reaching a maximum, declined to a lower steady-state level. Adding extracellular Ca²⁺ after thrombin caused an immediate further increase in Ca²⁺_cyt, resulting from Ca²⁺ influx (Figure 1A and 1B). The amplitude of the maximal Ca²⁺ rise, determined mainly by Ca²⁺ influx, depended on the period between the addition of thrombin and Ca²⁺. Figure 2A summarizes the time dependence of thrombin-induced Ca²⁺ influx, showing that it was maximal when Ca²⁺ was added 30 s after thrombin and that it decreased as the period between addition of thrombin and Ca²⁺ increased to 10 to 15 minutes. Ca²⁺ (1 mmol/L) added to unstimulated platelets resulted in only a minor increase in fura-2 ratio (Figure 2A), indicating that leakage of the dye out of platelets and passive thrombin-independent Ca²⁺ influx were both negligible. Thrombin also elicited Mn²⁺ influx, measured as the rate of quenching of fura-2 fluorescence (Figure 1C and 1D). Similar to Ca²⁺ influx, the rate of Mn²⁺ influx depended on the time after thrombin application. The Mn²⁺ influx was maximal after 30 s; however, after 5 minutes, thrombin caused almost no increase in the rate of Mn²⁺ influx as compared with the passive leak (Figure 2C).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of NO on thrombin-induced Ca2+ (A and B) and Mn2+ (C and D) influx. F340/F380 ratio (A and B) and F360 fluorescence (C and D; expressed as a percentage of F360 at the moment of Mn2+ addition) show the changes in Ca2+cyt and Mn2+ influx, respectively. Platelets were stimulated with thrombin (0.5 U/mL) in Ca2+-free medium followed by the addition of Ca2+ (1 mmol/L; A and B) or Mn2+ (100 µmol/L; C and D) 1 minute (A and C) or 10 minutes (B and D) after thrombin application (control traces). NO (1 µmol/L) was applied 30 s before Ca2+ or Mn2+ addition (+NO traces). In A and B, NO was also applied to the control platelets at the time shown after Ca2+ influx reached a plateau.

View larger version (43K): [in this window] [in a new window]   Figure 2. Summary data from experiments illustrated in Figures 1 and 3 showing the time dependence of thrombin- and BHQ-induced Ca2+ and Mn2+ influx and the effect of NO. Data represent the maximal rise in Ca2+ after its addition to the extracellular medium (A and B) and the initial rate of Mn2+ influx (C and D) at different times after application of thrombin (A and C) or BHQ (B and D) in control cells (open bars), in the presence of NO (1 µmol/L added 30 s before Ca2+ or Mn2+, solid bars), and in unstimulated cells (crosshatched bars). Each bar is an average±SEM of 3 to 7 experiments. *P<0.01; **P<0.001.

BHQ-Induced Cation Influx
In Ca²⁺-free medium, BHQ caused a slower, smaller, and more sustained increase in Ca²⁺_cyt (Figure 3B) compared with that of thrombin. Subsequent addition of external Ca²⁺ caused a rapid increase in Ca²⁺_cyt which, after reaching a peak, declined to a steady-state level (Figure 3A and 3B). Unlike the thrombin-activated Ca²⁺ influx, the amplitude of the BHQ-induced Ca²⁺ influx increased with time following application of BHQ (Figures 2B and 3), reaching a maximum after 5 to 10 minutes.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of NO on BHQ-induced Ca2+ (A and B) and Mn2+ (C and D) influx. Experiments are similar to those in Figure 1, but BHQ (30 µmol/L) was added to platelets instead of thrombin.

As with Ca²⁺ influx, BHQ-induced Mn²⁺ influx increased during the period between the addition of BHQ and Mn²⁺, being negligible during the first 30 s and reaching a maximum in 5 minutes. Figure 2D summarizes the relationship between the length of the period between addition of BHQ and Mn²⁺ and the rate of influx.

Effect of NO on Thrombin- and BHQ-Activated Ca²⁺ and Mn²⁺ Influx
To determine whether authentic NO has an inhibitory effect on capacitative cation influx in human platelets, NO was applied 30 s before addition of Ca²⁺ or Mn²⁺ in platelets stimulated with thrombin or BHQ. When added after thrombin-induced Ca²⁺ mobilization had reached the peak, NO (1 µmol/L) significantly increased the initial rate of decline in Ca²⁺_cyt (from 0.007±0.002 ratio units/s before NO addition to 0.088±0.004 ratio units/s after NO addition; n=6, P<0.001) (Figure 1A; see also Figure 4C) and profoundly inhibited the subsequent Ca²⁺ rise and Mn²⁺ influx (Figure 1). The effect of NO did not depend on the time of incubation with thrombin (Figure 2A and 2C), decreasing Ca²⁺ concentration and Mn²⁺ influx to a level that approximated the level that could be attributed to passive Ca²⁺ leak. When added to the thrombin-stimulated cells after Ca²⁺ influx reached equilibrium, NO decreased Ca²⁺_cyt to a level similar to that reached when NO was added before Ca²⁺ (Figure 1A and 1B). When NO-containing solution was exposed to the air for 1 hour and then applied to platelets, it produced no effect on Ca²⁺_cyt (not shown, n=3), indicating that NO, rather than the products of its degradation, was the active inhibitor of thrombin-induced cation influx.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of NO on the refilling of ionomycin-releasable Ca2+ stores. In the absence of extracellular Ca2+, ionomycin (2 µmol/L) was applied alone (A, Iono trace) or 5 minutes after thrombin (0.5 U/mL) (A through D, control trace) to release all stored Ca2+. NO (1 µmol/L) was added 30 s before (B, +NO trace), 1 minute after (C, +NO trace), or 4.5 minutes after thrombin (D, +NO trace).

Contrary to that induced by thrombin, the influx of Ca²⁺ and Mn²⁺ induced by BHQ was affected by NO only during the first few minutes of BHQ treatment (Figure 3A and 3C). Summary data are shown in Figure 2B and 2D. However, after pretreatment of platelets with BHQ for 5 to 10 minutes, NO did not affect the subsequent Ca²⁺ and Mn²⁺ influx (Figures 2B and 2D and 3B and 3D) significantly. These results illustrate the time-dependent disappearance of the effect of NO on capacitative cation influx during the progressive inhibition of SERCA by BHQ. This points to the importance of SERCA-dependent store refilling for NO-induced inhibition of influx.

NO Increases the Amount of Ca²⁺ Released by Ionomycin in Thrombin-Activated Platelets
We conducted the following experiments to determine whether NO affects Ca²⁺ stores in thrombin-activated platelets. Figure 4 shows thrombin-induced Ca²⁺ release in the absence of extracellular Ca²⁺. Five minutes after thrombin application, ionomycin (2 µmol/L) was applied to release the remaining portion of Ca²⁺ from the stores. When ionomycin was applied after thrombin, it caused a 2.5 times smaller rise in Ca²⁺_cyt than when it was applied alone to unstimulated platelets (Figure 4A; peak R_340/380 was 1.4±0.1 versus 3.5±0.3 ratio units, respectively; n=4, P<0.005). This indicates that after release from the stores by thrombin, Ca²⁺ is taken up into the stores and is also extruded from the cells. To test whether NO can increase the amount of Ca²⁺ in the stores after they were emptied by thrombin, NO (1 µmol/L) was added at different times before or after the agonist. When NO was applied 30 s before thrombin (Figure 4B), the amplitude of the thrombin response was slightly suppressed (peak R_340/380 was 1.0±0.1 versus 1.3±0.1 ratio units in control; n=4, P>0.05), but the initial rate of decline in Ca²⁺_cyt was significantly accelerated (from 0.007±0.001 to 0.014±0.001 ratio units/s; n=4, P<0.005). The amplitude of the ionomycin-induced Ca²⁺ release in this case was 65% more than that obtained without NO (peak R_340/380 was 2.2±0.1 compared with 1.4±0.1 ratio units without NO; n=4, P<0.001). When added 1 minute after thrombin, NO greatly accelerated the decline in Ca²⁺_cyt and increased the response to ionomycin by 36% (peak R_340/380 was 1.7±0.1 ratio units; n=4, P<0.05; Figure 4C). When NO was applied 30 s before ionomycin, it did not significantly increase the amplitude of Ca²⁺ release by ionomycin (peak R_340/380 was 1.5±0.1 ratio units; n=4, P>0.05; Figure 4D).

These results suggest that after Ca²⁺ is released from the stores by thrombin, NO can promote its uptake into the stores, which can be a result of acceleration of Ca²⁺ back-sequestration or, alternatively, inhibition of Ca²⁺ release. To discriminate between these possibilities, the following experiments were performed.

Inhibition of SERCA Abolishes the Effect of NO on Thrombin-Induced Ca²⁺ Release
In Ca²⁺-free medium, thrombin (0.5 U/mL) caused a fast Ca²⁺ rise resulting from Ca²⁺ release from the stores, followed by a slower Ca²⁺ decline as a result of its uptake into the stores and extrusion from the cell (Figure 5A). Addition of NO (1 µmol/L) 30 s before thrombin (Figure 5A) decreased the peak of transient Ca²⁺ rise (from 1.4±0.1 ratio units to 0.9±0.1; n=5, P<0.05) and dramatically accelerated the uptake/extrusion phase of Ca²⁺ transient (0.014±0.002 ratio units/s in the presence of NO versus 0.008±0.001 in control; n=5 P<0.05). When platelets were pretreated with 30 µmol/L BHQ for 5 minutes, Ca²⁺ was partially released from the stores, and thrombin was applied after BHQ released the remaining Ca²⁺ (Figure 5B). Pretreatment with BHQ eliminated the effect of NO on both peak thrombin–induced Ca²⁺ release (0.9±0.1 ratio units in control versus 0.9±0.1 in the presence of NO; n=5, P>0.05) and the rate of the subsequent uptake/extrusion (0.008±0.001 ratio units/s versus 0.008±0.0011; n=5, P>0.05). These experiments show that functional SERCA is required for the effect of NO on both rising and falling phases of thrombin-induced Ca²⁺ transient.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of BHQ on the inhibition of thrombin-induced Ca2+ mobilization by NO. Thrombin (0.5 U/mL) was applied in Ca2+-free medium in the absence (control traces) or presence (+NO traces) of NO (1 µmol/L applied at the time indicated) to the control platelets (A) or to the platelets in the presence of 30 µmol/L BHQ (B).

Inhibition of Mitochondria Does Not Prevent the Effect of NO on Ca²⁺_cyt
By taking up Ca²⁺ during agonist-induced activation, mitochondria could be involved in the regulation of capacitative Ca²⁺ influx in different types of cells.^{21 22 23} Inhibitors of mitochondrial metabolism were therefore used to determine whether mitochondria are involved in the inhibition of the thrombin-induced Ca²⁺ rise in intact platelets caused by NO. Figures 6A and 6B illustrate the effect of NO on thrombin-activated Ca²⁺ release and influx under control conditions (Figure 6A) and in the presence of the mitochondrial inhibitors (Figure 6B) oligomycin (1 µg/mL) and antimycin (2 µg/mL). As in the data obtained in the Jurkat cell line,²⁴ inhibition of mitochondria decreased the amplitude of thrombin-induced Ca²⁺ rise (from 1.8±0.2 to 1.4±0.1; n=3, P<0.05). However, the inhibition by NO of the thrombin-initiated Ca²⁺ release and influx was unchanged. These results indicate that uptake of Ca²⁺ into mitochondria is not involved in the inhibitory effect of NO on Ca²⁺_cyt in platelets.

View larger version (20K): [in this window] [in a new window]   Figure 6. Effects of inhibitors of mitochondria (A and B) or soluble guanylate cyclase (C and D) on the NO-induced inhibition of Ca2+ influx and acceleration of Ca2+ removal from the cytoplasm. Thrombin (0.5 U/mL) was applied in the absence of extracellular Ca2+, and then Ca2+ (1 mmol/L) was added to evoke Ca2+ influx in control platelets (A) or in platelets pretreated with oligomycin (1 µg/mL) and antimycin (2 µg/mL) for 2 minutes (B; control traces). Platelets were preincubated for 1 hour with 0.1% DMSO (C) or with 10 µmol/L ODQ (D). NO (1 µmol/L) was applied as shown after thrombin (+NO traces).

Inhibitor of cGMP Production, H-(1,2,4) Oxadiazolo(4,3-a) Quinoxallin-1-one (ODQ), Attenuates but Does Not Prevent the Effect of NO on Ca²⁺ Influx
To evaluate the role of cGMP formation in the effect of NO on Ca²⁺ influx, specific guanylate cyclase inhibitor, ODQ,^{25 26} was used. At the concentration used in these experiments, ODQ has been shown to completely prevent the NO-induced cGMP rise in platelets.²⁷ The effect of NO (1 µmol/L) on the rate of Ca²⁺ removal from the cytoplasm and on Ca²⁺ influx was compared in control platelets (preincubated with the vehicle, 0.1% DMSO) and in the platelets preincubated with ODQ (10 µmol/L) for 1 hour (Figure 6C and 6D). Pretreatment with ODQ did not alter the kinetics of the Ca²⁺-release phase or amplitude of the maximal Ca²⁺ influx following platelet stimulation with thrombin. However, it resulted in a small but significant attenuation of the inhibitory effect of NO on the Ca²⁺ increase associated with Ca²⁺ influx. The rates of the NO-induced acceleration of the falling phase of Ca²⁺ transient in control and ODQ-pretreated platelets were 0.051±0.003 ratio units/s and 0.039±0.003, respectively (n=5, P<0.05), which corresponds to a 24% decrease. The amplitude of the thrombin-induced Ca²⁺ influx phase was inhibited by NO by 82% in the cells preincubated with vehicle (1.46±0.17 ratio units versus 0.26±0.02; n=5, P<0.01) and only by 67% in ODQ-treated cells (1.44±0.16 ratio units versus 0.47±0.06; n=5, P<0.01). Thus, the effect of NO (1 µmol/L) on Ca²⁺ influx in platelets is partially mediated by cGMP formation. These experiments also indicate the possible existence of another, cGMP-independent mechanism of NO-induced inhibition of capacitative cation influx in platelets.

NO Accelerates Ca²⁺ Uptake by the Stores in Permeabilized Platelets
To directly monitor the process of filling and emptying of internal Ca²⁺ stores and to eliminate the involvement of the IP₃ cascade and plasma membrane-associated regulatory proteins (plasma membrane Ca²⁺-ATPase [PMCA] or Na⁺/Ca²⁺ exchanger), the free-Ca²⁺ level was measured in suspensions of permeabilized platelets. Addition of saponin (20 µg/mL) to the platelets resuspended in cytoplasmic solution caused an immediate decrease in free Ca²⁺ (Figure 7A), which was completely prevented by BHQ (30 µmol/L) added 2 minutes before saponin (not shown). In the presence of 2 mmol/L ATP, cytoplasmic Ca²⁺ decreased from 1 to 3 µmol/L to a stable level or set point^{28 29} of 200 to 250 nmol/L and remained at the same low level for at least 10 minutes. When Ca²⁺ (1 µmol/L) was added to permeabilized cells, it was rapidly taken up by the stores, and the same level of free Ca²⁺ was restored (Figure 7A). This decrease in free Ca²⁺ concentration was used in our experiments as a measure of Ca²⁺ uptake by the stores. Application of IP₃ (250 µmol/L) caused the rapid release of stored Ca²⁺, which also was almost completely resequestered into the stores. Inhibition of SERCA with BHQ caused slow and irreversible release of Ca²⁺ from the stores into the cytoplasmic solution. Heparin, a blocker of IP₃ receptors,^{30 31} completely abolished IP₃-induced Ca²⁺ release but was without any effect on the Ca²⁺ rise and uptake after exogenously added Ca²⁺ (Figure 7B), indicating that these processes do not involve IP₃-sensitive channels.

NO (1 µmol/L) significantly increased the rate of Ca²⁺ uptake after IP₃-induced release (0.077±0.006 versus 0.024±0.003 ratio units/s in control; n=5, P<0.001; Figure 7C), but it had no effect if applied after BHQ (Figure 7D). Because the effect of NO on IP₃-induced Ca²⁺ release could be due either to inhibition of IP₃-induced Ca²⁺ release or to acceleration of uptake into the stores, the effect of NO on Ca²⁺ sequestration in the absence of IP₃ was determined.

Exogenous Ca²⁺ was added to the suspension of platelets after the cells were permeabilized and the set point was allowed to reestablish. The rate of uptake of exogenously added Ca²⁺, as well as the lowest free-Ca²⁺ level reached after Ca²⁺ addition and uptake, was highly dependent on the ATP concentration (Figure 8A and 8B). When added 30 s before Ca²⁺, NO significantly accelerated the rate of Ca²⁺ sequestration into the stores (Figure 8C). However, the effect of NO was strongly dependent on the rate of sequestration observed in the absence of NO, which, in turn, depended on ATP concentration (Figure 8C and 8D). For example, at the higher rate of Ca²⁺ uptake typical for 2 mmol/L ATP (Figure 8B), there was no effect of NO (Figure 8D; 0.042±0.005 ratio units/s under control conditions and 0.044±0.006 ratio units/s in the presence of NO; n=4, P>0.1). However, at lower ATP concentrations (100 to 200 µmol/L ATP), NO significantly accelerated Ca²⁺ uptake (Figure 8C), but this was observed only in those experiments in which the control rate was lower than 0.03 ratio units/s (Figure 8E). For these experiments, NO significantly increased the rate of Ca²⁺ uptake into the stores from 0.015±0.003 in control to 0.022±0.003 ratio units/s in the presence of NO (n=7, P<0.05), representing an average 47% increase. Figure 8E summarizes the dependence of the effect of NO on the control rate of Ca²⁺ uptake measured in platelets from different platelet donors at 3 different ATP concentrations (100 µmol/L, 200 µmol/L, and 2 mmol/L). At low concentrations of ATP, when only part of the added Ca²⁺ was taken into the stores (Figure 8A and 8B), NO also increased the percentage of Ca²⁺ uptake from 49±11 to 68±8 (n=7, P<0.01). Thus, NO was able to increase the rate and quantity of Ca²⁺ uptake into the stores, but only when the uptake was submaximal under our experimental conditions.

View larger version (26K): [in this window] [in a new window]   Figure 8. NO-induced acceleration of Ca2+ uptake in permeabilized platelets. A, Ca2+ transient (normalized to the maximal Ca2+ rise) following addition of Ca2+ (1 µmol/L) in the presence of 500 and 50 µmol/L ATP. B, The dependence on ATP concentration of the rate of initial Ca2+ uptake (left scale) and the percentage of maximal Ca2+ uptake (right scale). The rate of initial Ca2+ uptake was estimated by linear fit during the first 20 s of the decline in free Ca2+ in experiments such as the one shown in panel A. C and D, The decrease in Ca2+ (normalized to the maximum rise) following application of external Ca2+ (1 µmol/L) in the presence of 200 µmol/L ATP (C) or 2 mmol/L ATP (D) in the absence of NO (control) or presence of NO (+NO, 1 µmol/L added 30 s before Ca2+). Dotted lines show the linear fit of the initial Ca2+ uptake in the presence or absence of NO. E, The relationship between the initial rate of Ca2+ uptake under control conditions at 100 µmol/L (), 200 µmol/L (), or 2 mmol/L () ATP and the uptake after addition of 1 µmol/L NO. One hundred percent is the rate of Ca2+ uptake in the absence of NO. Each point represents a single experiment such as the one shown in panel C or panel D. The arrow indicates experiment shown in panel C.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, we showed that NO indirectly inhibits store-operated cation influx in vascular smooth muscle cells and proposed a hypothesis that this inhibition is a result of NO-induced acceleration of Ca²⁺ back-sequestration into the stores.³² Here we tested this idea in human platelets and provided new evidence showing that NO inhibits capacitative cation influx by accelerating the refilling of the stores, predominantly through increased SERCA-dependent Ca²⁺ uptake.

Nonselective Cation Influx in Platelets Depends on the Stores
Our data are consistent with a close correlation of cation influx in human platelets with the filling state of the stores.^{4 5 6} Thrombin causes a fast release of Ca²⁺ from IP₃-sensitive Ca²⁺ stores in platelets, which is followed by partial back-sequestration into the stores and Ca²⁺ extrusion from cells via PMCA and Na⁺/Ca²⁺ exchanger.³³ Under our experimental conditions Ca²⁺ and Mn²⁺ influx is maximally activated during the first 30 s after thrombin application and then gradually decreases. This decrease most probably results from the time-dependent partial back-sequestration of Ca²⁺ into the stores. However, we cannot rule out the involvement of other possible reasons, including desensitization of the thrombin receptor signaling cascade or inactivation of plasma membrane cation channels.

When Ca²⁺ was released slowly with BHQ (as was seen to occur in permeabilized platelets in Figure 7D), the amplitude of Ca²⁺ influx as well as the rate of Mn²⁺ influx increased gradually with time, reaching a maximum after 5 minutes (Figure 2B and 2D). The absence of back-sequestration of Ca²⁺ in the presence of BHQ might explain why the maximal amplitude of the BHQ-induced Ca²⁺ rise was 2.5 times higher than that induced by thrombin (Figure 2A and 2B). The rates of Mn²⁺ influx in thrombin- and BHQ-treated platelets were comparable (Figure 2C and 2D), suggesting that SERCA-dependent Ca²⁺ back-sequestration into the stores in the presence of thrombin, which is absent in BHQ-treated cells, can effectively reduce Ca²⁺ influx. Thus, the maximal Ca²⁺ and Mn²⁺ influx in platelets coincides with the maximal emptying of the stores, consistent with a mechanism of capacitative cation influx.

NO Inhibits Capacitative Cation Influx in Human Platelets, but SERCA-Dependent Store Refilling Is Required
Our data indicate that SERCA-dependent refilling of the stores is required for NO to inhibit Ca²⁺ and Mn²⁺ influx. When Ca²⁺/Mn²⁺ influx was activated by thrombin, NO was very effective and blocked this influx almost completely and independently of the time after thrombin application (Figures 1 and 2). In BHQ-treated platelets, however, NO was able to partially inhibit Ca²⁺ and Mn²⁺ influx and the rise in Ca²⁺_cyt only during the first few minutes after BHQ application. During this initial phase, presumably, SERCA is only partially inhibited and some back-sequestration of Ca²⁺ into the stores can still occur. After 5 to 10 minutes, when the process of SERCA inhibition by BHQ is apparently complete and maximal cation influx has developed (Figure 2), the inhibitory effect of NO on Ca²⁺_cyt disappears. The absence of the inhibitory effect of NO on maximum Ca²⁺ and Mn²⁺ influx after a 10-minute treatment with BHQ suggests that NO does not have a direct effect on plasma membrane cation channels, which are responsible for such influx.

The time-dependent loss of the effect of NO on cation influx demonstrated in the presence of the SERCA inhibitor could explain why some researchers^{18 34} failed to detect the inhibition of TG-induced cation influx in platelets by NO donors, while others¹⁷ reported such an inhibition. The variability in the effect of NO could be also related to the incomplete inhibition of all SERCA isoforms by TG under different experimental conditions or to the differences between BHQ- and TG-sensitive Ca²⁺ stores and their role in the regulation of cation channels.

NO Promotes SERCA-Dependent Back-Sequestration of Ca²⁺ Into the Stores
The absence of an effect of NO on capacitative cation influx when SERCA is inhibited shows that NO does not inhibit the influx directly and suggests the importance of functional SERCA for the NO-induced inhibition of cation influx. To determine whether NO indeed can accelerate the refilling of Ca²⁺ stores in a SERCA-dependent manner, the following 2 approaches were used. In the first approach, ionomycin was used at a high concentration to release Ca²⁺ from all the stores to determine whether NO can promote the refilling of the stores emptied by thrombin. In thrombin-activated platelets, NO was shown to significantly increase the total intracellular Ca²⁺ pool released by ionomycin. The effect of NO was maximal if it was added before initiation of Ca²⁺ release by thrombin, apparently because it promotes back-sequestration of Ca²⁺ released by IP₃. When NO was added later, most of the released Ca²⁺ was already removed from the cytoplasm, which explains the insignificant increase in the Ca²⁺ stores (Figure 4).

Permeabilized platelets were used in the second approach, which allows direct observations of the Ca²⁺ movements in and out of intracellular stores. Using this method, NO was found to accelerate uptake of Ca²⁺ either after it was released from the stores with IP₃ or after exogenous Ca²⁺ was added (Figure 8). The effect of NO disappeared in the presence of BHQ. The strong dependence on ATP concentration of Ca²⁺ uptake in permeabilized platelets (Figure 8) points to the involvement of an ATP-using process, which, most logically, is SERCA. Importantly, the effect of NO strongly depends on the initial rate of Ca²⁺ uptake (Figure 8). At a maximal rate of Ca²⁺ sequestration, the possibility of a further increase of SERCA activity by NO is unlikely. Under physiological conditions, one would expect SERCA to work at a lower, submaximal rate that would allow its further acceleration by NO. In permeabilized platelets, NO had a stimulatory effect only when the rate of Ca²⁺ uptake was experimentally decreased (by decreasing the concentration of ATP) (Figure 8C and 8E). These results provide a possible explanation of the fact that some investigators did not observe an effect of NO on SERCA.³⁵ Indeed, SERCA activity is usually measured under experimental conditions providing its highest activity (at high Ca²⁺ and/or ATP concentrations), which might exclude its further acceleration by NO.

Role of IP₃-Induced Ca²⁺ Release, PMCA, Na⁺/Ca²⁺ Exchanger, and Mitochondria in the Effect of NO on Ca²⁺_cyt in Human Platelets
NO has been shown to inhibit the transient Ca²⁺ rise caused by agonists in a variety of cells, including platelets,^{14 16} which theoretically can be mediated through NO-induced inhibition of IP₃ production by phospholipase C or suppression of IP₃-induced Ca²⁺ release, on one hand, and a rapid acceleration of Ca²⁺ back-sequestration into the stores, on the other hand.

Although we cannot completely exclude an effect of NO exerted by changes in the production or action of IP₃, there are several reasons why back-sequestration of Ca²⁺ into the stores by SERCA appears to be the major physiological mechanism for the NO-induced decrease in Ca²⁺_cyt in human platelets. First, inhibition of SERCA with BHQ (which prevents Ca²⁺ back-sequestration, but not IP₃-induced Ca²⁺ release) completely abolished the effect of NO on the thrombin-induced transient Ca²⁺ rise (Figure 5). Second, even without involvement of IP₃ in permeabilized platelets, NO significantly increased uptake of externally added Ca²⁺, accelerating the rate of uptake and lowering the free Ca²⁺ level (Figure 8). Third, the effect of NO depended on ATP concentration (Figure 8), pointing to the regulation of an ATP-dependent process such as SERCA.

PMCA and Na⁺/Ca²⁺ exchanger have been proposed to be targets for NO action in other cells (for review, see Reference 36³⁶ ). In human platelets, we found no evidence for a significant role of these Ca²⁺-removal mechanisms in the effects of NO. Indeed, when SERCA was inhibited by BHQ, Ca²⁺ (released by thrombin) was successfully removed from the cytoplasm (Figure 5), pointing to the activity of PMCA, Na⁺/Ca²⁺ exchanger, or both. However, NO did not affect Ca²⁺ removal when SERCA was inhibited. Also, there was no effect of NO on the peak Ca²⁺ rise in BHQ-treated platelets, although such extrusion mechanisms as PMCA and Na⁺/Ca²⁺ exchanger are expected to work under these conditions.

By taking up Ca²⁺ during agonist-induced activation, mitochondria could be involved in the regulation of capacitative Ca²⁺ influx in different types of cells.^{21 22 23} Our experiments with mitochondrial inhibitors (Figure 6) show that the effect of NO on intracellular Ca²⁺ does not depend on oxidative metabolism by, or Ca²⁺ uptake into, mitochondria.

Thus, IP₃-induced Ca²⁺ release, PMCA, Na⁺-Ca²⁺ exchanger, and mitochondria, although they are potential targets for NO, seem not to play an important role in the effects of NO on Ca²⁺_cyt in human platelets.

Role of cGMP Production in the Effect of NO on Ca²⁺ Influx in Human Platelets
It is well known that NO³⁷ and NO donors^{27 38} stimulate cGMP production in human platelets leading to the activation of cGMP-dependent protein kinase and inhibition of agonist-induced Ca²⁺_cyt rise and platelet aggregation.^{37 39} In rat aortic smooth muscle cells, this effect was partially attributed to the phosphorylation of the SERCA-regulatory protein phospholamban and thus the increased sensitivity of SERCA to Ca²⁺.⁴⁰ It is tempting to look for a similar explanation for the effect of NO in platelets. In fact, a phospholamban-like 22-kDa protein, thrombolamban, found in platelets,⁴¹ has been shown to regulate the activity of Ca²⁺ uptake by the ER vesicles. However, comparative analysis of phospholamban and thrombolamban revealed differences in their structural and physical properties, as well as in the mechanisms for activation of the respective Ca²⁺ pumps.⁴² Moreover, no phosphorylation of thrombolamban by sodium nitroprusside or cGMP analogs was found in platelets.^{39 43} Thus, it seems unlikely that cGMP-dependent stimulation of platelet SERCA by NO is mediated by thrombolamban.

Inhibition of guanylate cyclase by ODQ has been shown to eliminate the cGMP rise caused by NO donors and to prevent their effect on platelet aggregation.²⁷ However, there are some indications that cGMP might not account for all the effects of authentic NO on Ca²⁺_cyt. It was shown recently that inhibition of cGMP production with ODQ suppresses, but does not eliminate, the effects of authentic NO on intracellular Ca²⁺ in smooth muscle cells,⁴⁴ as well as the effect of the NO donor S-nitrosoglutathione in platelets.⁴⁵ Similarly, we found that pretreatment of human platelets with 10 µmol/L ODQ for 1 hour only partially blocked the inhibitory effect of NO on capacitative Ca²⁺ influx (Figure 6). This result points to the possibility that along with a cGMP-dependent pathway there is also another, cGMP-independent mechanism of NO effect on Ca²⁺ influx in human platelets. Indeed, direct NO-induced nitrosylation of critical thiols is known to be responsible for cGMP-independent effects of NO on some other proteins.^{46 47 48} Further studies of the effects of NO on SERCA need to be done to fully understand this physiologically important pathway for regulation of capacitative cation influx and intracellular Ca²⁺ in platelets and other nonexcitable cells.

	Acknowledgments

 
This work was supported by National Institutes of Health grants Specialized Center of Research HL55993 and R01 HL54150. We would like to thank Dr E. Simons for help in establishing the platelet isolation procedures and Dr B. Corkey for useful suggestions and comments on the experiments with permeabilized platelets.

Received July 17, 1998; accepted November 19, 1998.

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