Calcium adaptation to sodium pump inhibition in a human megakaryocytic cell line

Masayuki Kimura, Xiaojian Cao, and Abraham Aviv

Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

Submitted 24 February 2005 ; accepted in final form 25 May 2005


    ABSTRACT
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 ABSTRACT
 METHODS
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The unique characteristics of the platelet Na/Ca exchanger, i.e., its dependence on both transmembrane Na and K gradients, render it highly sensitive to Na pump inhibition. In this project, we observed that the human megakaryocytic cell line CHRF-288 expresses both the {alpha}1- and {alpha}3-isoforms of the Na-K-ATPase. Inhibition of the Na pump increased the RNA and protein expressions of sarco(endo)plasmic reticulum Ca-ATPase 2b, cytosolic Na and Ca, and the freely exchangeable Ca in the endoplasmic reticulum. These changes occurred in concert with diminished store-operated Ca entry and an increase in the maximal activity of the Na/Ca exchanger. Inhibition of the Na pump by ouabain was more effective in inducing these changes than diminishing medium K. Collectively, these observations point to an integrative effort to counteract the impact of Na pump inhibition by Ca sequestration into the endoplasmic reticulum, diminished Ca entry, and increased activity of the Na/Ca exchanger. The implications of these findings in platelet biology are discussed.

platelets; thrombosis; potassium; stroke


CYTOSOLIC CALCIUM (Cac) is critical for platelet activation because it regulates an array of Ca-dependent platelet enzymes (21). Platelet Cac regulation is controlled by Ca fluxes across the plasma membrane and Ca sequestration into and egress from intracellular organelles, particularly the dense tubules, which are analogous to the sarco(endo)plasmic reticulum (SER) in nucleated cells. The tight regulation of platelet Cac is crucial, as a host of agonists activate platelets through Ca signaling (21). To better understand platelet Cac regulation and its relationship to platelet function, our previous research (12, 14) has focused on the platelet Na/Ca exchanger (NCX). We found that, unlike the more ubiquitous cardiac NCX, which is driven by the Na gradient across the plasma membrane (16, 20), the platelet NCX is driven by both the transmembrane Na and K gradients. In addition, we found that the platelet NCX is in fact the retinal rod NCX (14).

We suggest that the dependence of the platelet NCX on both the Na and K gradients across the plasma membrane heightens platelet sensitivity to perturbations in plasma Na/K levels and particularly to circulating inhibitors of the Na pump. Such inhibitors not only raise cytosolic Na (Nac) but also lower cytosolic K (Kc); the joint effect of these perturbations would suppress Ca extrusion from platelets by the NCX. Moreover,the high sensitivity of platelets to Na pump inhibitors also may arise from their rudimentary ability to counteract the inhibition of the Na pump through nucleus-mediated adaptive responses, given that platelets are anucleated cells. Thus circulating platelets may be highly sensitive to acute fluctuations in systemic Na/K status, presumably through factors regulating the Na pump.

To understand the potential impact of megakaryocytic adaptation to Na pump inhibition, we used CHRF-288, a human megakaryocytic cell line (6), to explore 1) the nature of the {alpha}-subunit isoforms of the Na pump (Na-K-ATPase) in megakaryocytes and 2) the effect of Na-K-ATPase inhibition for 1–4 days on the protein expression of SER Ca-ATPase (SERCA) 2b, Nac, Cac, the freely exchangeable Ca (FECa) in the SER, the activity of store-operated Ca entry (SOCE), and the activity of the NCX. The conclusion we draw from the findings is that megakaryocytes upregulate the SERCA, increase their NCX activity, and diminish SOCE to attenuate the impact of Na pump inhibition.


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Cell culture. Cells from a human megakaryocytic cell line, CHRF-288, were provided by G. W. Dorn II and M. Lieberman (University of Cincinnati, Cincinnati, OH). The cells were cultivated at 37°C, 5% CO2-95% air in Fisher's medium supplemented with 10% fetal BSA and 100 U/ml penicillin G plus 100 µg/ml streptomycin. Cells were passed by dilution to ~0.5 x 108 cells/ml in fresh medium every 3–4 days.

Cytosolic Ca and cytosolic Na. In preparation for measurements of Cac, CHRF-288 cells were washed twice with HEPES buffer consisting of (in mmol/l) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose plus 0.1% BSA. Cells were incubated for 30 min at 37°C with 5 µmol/l fura-2 AM (Molecular Probes, Eugene, OR). The extracellular dye was removed by centrifugation. Cac measurement was performed at 37°C under constant stirring in SPEX Fluoromax (Edison, NJ). These measurements were initiated within 30 s of resuspending the cells in either 1 mmol/l CaCl2 or Ca-free HEPES buffer (0.3 mmol/l EGTA added). Excitation wavelengths were set at 340 and 380 nm, and emission wavelength was set at 505 nm. Maximum and minimum fluorescence ratios were determined by the addition of 20 µmol/l digitonin in the presence of 1 mmol/l CaCl2, followed by 10 mmol/l EGTA (final pH 8.5). Autofluorescence was determined at the end of each experiment by the addition of 1 mmol/l MnCl2 and 20 µmol/l digitonin.

For Nac measurements, CHRF-288 cells were washed as above and incubated in a HEPES buffer with 10 µmol/l Na-binding benzofuran isophthalate (SBFI)-AM and 20% (wt/vol) Pluronic F-127 for 1 h at 37°C. Excitation wavelengths were set at 340 and 385 nm, and emission wavelength was set at 505 nm. Calibration of Nac was accomplished by the gramicidin method as previously described (23). Gramicidin D (2 µmol/l), monensin (10 µmol/l), and nigericin (10 µmol/l) were added to calibration solutions consisting of (in mmol/l) 0–115 Na gluconate, 0–115 K gluconate, 30 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.4). The ratios of fluorescence intensities at eight different concentrations of Nac were used to obtain the standard parameters. Autofluorescence was obtained with unloaded cells.

FECa in SER. The rapid increase in Cac of fura-2-loaded CHRF-288 cells in response to 5 µmol/l ionomycin and 500 nmol/l thapsigargin (to inhibit Ca reuptake by the SER) in Ca-free HEPES buffer was used as an indicator of FECa in the SER (illustrated in Fig. 1).



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Fig. 1. A composite of 3 traces demonstrating the way in which freely exchangeable Ca (FECa) is measured. Ouabain cells were grown in the presence of 25 nmol/l ouabain in the growth medium for 4 days. Control cells were grown in the presence of the vehicle (DMSO). Arrow indicates addition of ionomycin and thapsigargin to cells suspended in a Ca-free buffer. Basal cytosolic Ca (Cac; monitored by fura-2) is subtracted from the peak Cac signal to obtain the FECa. Vertical bars denote SE.

 
NCX activity and SOCE measurements. Washed CHRF-288 cells were resuspended in Ca-free HEPES buffer consisting of (in mmol/l) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 0.3 EGTA (pH 7.4). Cells were incubated with 0.1 mmol/l thapsigargin for 60 min, and 5 µmol/l fura-2 AM was added for the last 30 min. Cells were centrifuged and resuspended in Na loading buffer consisting of (in mmol/l) 145 NaCl, 1 MgCl2, 10 HEPES, 10 glucose, and 0.3 EGTA (pH 7.4) plus 10 µmol/l monensin, 10 µmol/l nigericin, and 0.1 mmol/l ouabain. The cells were incubated for 10 min, and BSA (1%) was added to stop the reaction. Cells were centrifuged and resuspended in K buffer consisting of (in mmol/l) 145 KCl, 10 HEPES, 10 glucose, and 0.3 EGTA plus 0.1% BSA (pH 7.4) or Na buffer consisting of (in mmol/l) 145 NaCl, 10 HEPES, 10 glucose, and 0.3 EGTA plus 0.1% BSA (pH 7.4). To measure the NCX activity, 0.34 mmol/l CaCl2 (final 40 µmol/l) was added to K buffer, and the first 60-s increase in Cac was monitored. Under these conditions NCX activity is measured when the outward Na and inward K gradients are each 145 mmol/l (3). Figure 2A illustrates the K- and Na-dependent Ca entry (the NCX activity) on addition of CaCl2 to the medium at a final concentration of 40 µmol/l Ca. Under these circumstances, the rate of increase in Cac is the same with SKF-96365 (dissolved in DMSO), an inhibitor of SOCE, and without it (DMSO alone). Figure 2A underscores the fact that at a low concentration (40 µmol/l Ca) in the medium, there is no apparent entry of Ca through SOCE. We note that the measurements of NCX activity were performed under circumstances in which we artificially established the Na/K/Ca gradients across the CHRF-288 cells. These findings reflect NCX activity under uniform circumstances for both control cells and cells that had been grown in medium that inhibited the Na-K-ATPase.



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Fig. 2. Illustration of the methods and conditions for the measurements of the activity of Na/Ca exchange (NCX; reverse mode) and store-operated Ca entry (SOCE). Activity of NCX (A) was measured in a K buffer, so that the inward K and outward Na gradients were set at 145 mmol/l. Activity of SOCE (B) was measured in a Na buffer, when the inward and outward Na gradients were set at 145 mmol/l. Activities of both transport systems were measured as the 60-s increase in Cac after addition of Ca (arrows) to the buffer. The rise in Cac following the addition of low Ca (40 µmol/l final concentration) into K buffer represented NCX activity, because at this low external Ca concentration there was no evidence for SOCE (shown by lack of response to presence of 50 µmol/l SKF-96365) in the buffer (A). In contrast, the rise in Cac following the addition of high Ca (0.5 mmol/l final concentration) to a Na buffer was totally ablated in the presence of SKF-96365 in the buffer (B).

 
To measure the SOCE, CaCl2 at a final concentration of 0.5 mmol/l was added to Na buffer, and the first 60-s increase in Cac was monitored (Fig. 2B). Under these conditions, the increase in Cac was abolished in the presence of SKF-96365.

Western blot analysis. CHRE-288 cells were washed twice with buffer consisting of (in mmol/l) 140 NaCl, 5 KCl, 10 glucose, 3 EDTA, and 10 HEPES, plus 0.005 U/ml aprotinin and 20 µmol/l PMSF (pH 7.5). Washed cells were sonicated in a buffer consisting of (in mmol/l) 100 KCl, 15 NaCl, 12 sodium citrate, 2 MgSO4, 10 glucose, and 25 HEPES plus 0.2 mmol/l PMSF, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, 0.05 U/ml aprotinin, and 1 mmol/l dithiothreitol (pH 7.5). Thereafter, cells were centrifuged at 19,000 g for 25 min. The supernatant was further centrifuged at 100,000 g for 60 min. The pellet was resuspended in HEPES buffer. The protein content was measured by the Bio-Rad method. Cell membrane protein (10–15 µg/well) was electrophoresed on 6% or 7.5% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. After the nitrocellulose membrane was blocked with 5% milk in Tris-buffered saline (TBS; 150 mmol/l NaCl, 10 mmol/l Tris) for 30 min, nitrocellulose membranes were incubated with the corresponding primary antibodies (Abs) in 1% milk-TBS for 1 h. Nitrocellulose membranes were then washed three times in TBS-0.1% Tween 20 for 5 min, rinsed with TBS, and incubated with horseradish peroxidase-conjugated secondary Ab in 1% milk-TBS for 1 h. Nitrocellulose membranes were washed three times in TBS-0.1% Tween 20 and rinsed with TBS. The blots were developed with enhanced chemiluminescence (ECL, Amersham) and quantitated by densitometry (Molecular Dynamics, Computing Densitometer model 300B, Image Quant Version 3.3).

The following antibodies were used against the {alpha}-isoforms of the Na-K-ATPase: {alpha}1 (catalog no. 05-585) and {alpha}2 (catalog no. 06-168) from Upstate Biotechnology (Lake Placid, NY) and {alpha}3 (catalog no. MA3-915) from Affinity Bioreagents (Golden, CO). Anti-SERCA 2b was also from Affinity Bioreagents (catalog no. MA3-910).

RT-PCR. Total RNA from CHRF-288 cells was isolated with TRIzol (Invitrogen, Carlsbad, CA), and RT-PCR was performed with the One-Step RT-PCR System (Invitrogen). PCR product was analyzed on the 1% agarose gel. The following pairs of subunit-specific primers were generated: {alpha}1, 5'-GGCAGTGTTTCAGGCTAA-3' and 5'-TTCATCTGGCAGAAAGAGG-3' (expected product length = 400 bp); {alpha}2, 5'-TGGAGACCCGCAATATCTGT-3' and 5'-GTGTTCAATCTCCATTGCT-3' (expected product length = bp); {alpha}3, 5'-CTTGGAGACTCGGAACATCA-3' and 5'-CAAGCCAGGTGTATCCGAGA-3' (expected product length = 248 bp).

Northern blots. Total RNA (10 µg) was separated on 1% agarose gel in MOPS buffer (in mmol/l: 20 MOPS, 5 Na acetate, 1 EDTA, pH 7.0) and transferred onto nylon membrane. The membranes were prehybridized for 2 h at 65°C in solution H [50% formamide, 5x SSC, 0.1% sarkosyl, 2% SDS plus 5% (wt/vol) blocking reagent; Roche Applied Science, Indianapolis, IN] and hybridized overnight at 65°C in solution H with digoxigenin-labeled SERCA isoform-specific riboprobes. Northern blots were detected by chemiluminescent detection with CDP-Star (Roche Applied Science) and quantitated by densitometry.

Statistics. One-way ANOVA (with Duncan's multiple-range test for variable) was used for all statistical analyses. A P value <0.05 was considered statistically significant. Each experiment was performed on three different occasions.


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Expression of Na-K-ATPase isoforms. In the first set of experiments we examined expression profiles of isoforms of the {alpha}-subunit of the Na-K-ATPase, which contain the binding site for cardiac glycosides, to ascertain that ouabain exerts a specific effect in these cells. RT-PCR analysis revealed that CHRF-288 cells express both {alpha}1- and {alpha}3- but not {alpha}2-isoforms (Fig. 3A). Western blot analysis (Fig. 3B) confirmed these findings. We also confirmed the expressions of the {alpha}1- and {alpha}3-isoforms in platelets, although the expression of the {alpha}3-isoform was considerably less than that of the {alpha}1-isoform (not shown).



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Fig. 3. One of 3 runs illustrating presence of both the {alpha}1- and {alpha}3-subunit isoforms of Na-K-ATPase in CHRF-288 cells: RT-PCR (A) and Western blot (B) analyses for the {alpha}-subunits are shown. In A, 250 ng of total RNA was used. In B, 15 µg of protein was used per well. Cells were grown in a standard Fisher's medium without ouabain.

 
Na-K-ATPase inhibition and cell number. We used either ouabain or reduction of K in the growth medium to inhibit Na-K-ATPase in CHRF-288 cells. Figure 4A shows that there was no apparent effect of 6.25 and 12.5 nmol/l ouabain in the medium for 4 days on cell numbers, but there was a drop in cell numbers when 25 nmol/l ouabain was present for 4 days in the medium. At a fixed concentration of 25 nmol/l, ouabain exerted a slight but significant effect on cell numbers, an effect that increased with the duration of treatment (Fig. 4B). Diminishing the medium K concentration for 4 days from the basal level of 5 mmol/l to 0.5, 0.25, and 0.125 mmol/l also resulted in a progressive, although slight, decline in cell number (Fig. 4C). It should be noted that in a previous study (14) we did not find that 25 nmol/l ouabain caused a reduction in CHRF-288 cell number. The experiments described in that report were in CHRF-288 cells grown in 20% horse serum, whereas the present experiments were performed in cells grown in 10% FBS. The culture conditions may account for this difference between the studies.



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Fig. 4. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on cell number: dose response to presence of ouabain in the growth medium for 4 days (A), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B), and effect of lowering of medium K for 4 days (C) are shown. A: *significantly lower than the other conditions. B: *significantly lower than control (Con); {dagger}significantly lower than 1-day ouabain. C: *significantly lower than 5.0 mM KCl; {dagger}significantly lower than the other conditions.

 
Na-K-ATPase inhibition by ouabain and SERCA 2b expression. Platelets express both SERCA 2b and SERCA 3 isoforms. To monitor the response of SERCA to Na-K-ATPase inhibition in CHRF-288 cells, we followed the expression of SERCA 2b after 72-h treatment with ouabain (concentrations of 12.5 and 25 nmol/l in the growth medium). Figure 5, A and B, illustrates the RNA and protein expressions of SERCA 2b in control cells and cells treated with 25 nmol/l ouabain. Figure 5C depicts the effect of ouabain treatment on the protein expression of SERCA 2b. It is clear that inhibition of the Na pump upregulates SERCA 2b expression.



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Fig. 5. Ouabain upregulates sarco(endo)plasmic reticulum (SER) Ca-ATPase (SERCA) 2b expression in CHRF-288 cells: illustrations of a 3-day ouabain treatment on mRNA (A) and protein (B) expressions of SERCA 2b. Lanes 1 and 2 in A and B represent untreated and ouabain-treated cells, respectively. C: summary of 3 experiments, showing the effect of 2 doses of ouabain on SERCA 2b protein expression. *Significantly higher than Con.

 
Na-K-ATPase inhibition and Nac. Both ouabain addition (Fig. 6, A and B) and reduction of K in the growth medium increased Nac. We note that at concentrations of 6.25–25 nmol/l for 4 days, ouabain raised Nac in a dose-responsive fashion. The same held for reduction of K in the growth medium.



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Fig. 6. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on cytosolic Na (Nac): dose response of ouabain presence in the growth medium for 4 days (A), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B), and effect of lowering of medium K for 4 days (C) are shown. A: *significantly higher than Con and 6.25 nM ouabain; {dagger}significantly higher than the other conditions. B: *significantly higher than Con. C: *significantly higher than 5.0 mM KCl; {dagger}significantly higher than the other conditions.

 
Na-K-ATPase inhibition and Cac. Figure 7 depicts the effect of ouabain (Fig. 7, A, B, D, and E) and K reduction (Fig. 7, C and F) in the growth medium on Cac in CHRF-288 cells. Both modes of Na-K-ATPase inhibition raised Cac in a dose-responsive manner. The higher levels of Cac in Ca-containing HEPES buffer (Fig. 7, D–F) than in Ca-free buffer (Fig. 7, A–C) reflect a steady state that includes Ca fluxes from the external medium.



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Fig. 7. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on Cac. Measurements were performed in Ca-free medium (A–C) and in the presence of Ca (1 mmol/l) in the medium (D–F). Dose response to ouabain presence in the growth medium for 4 days (A and D), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B and E), and effect of lowering of medium K for 4 days (C and F) are shown. A: *significantly higher than the other conditions. B: *significantly higher than Con; {dagger}significantly higher than 1-day ouabain. C: *significantly higher than 5.0 mM KCl. D: *significantly higher than Con and 6.25 nM ouabain. E: *significantly higher than Con. F: *significantly higher than 5.0 mM KCl; {dagger}significantly higher than the other conditions.

 
Na-K-ATPase inhibition and FECa in SER. Whereas inhibition of Na-K-ATPase by ouabain considerably increased FECa in a dose-responsive (Fig. 8A) and time-responsive (Fig. 8B) manner, inhibition of the enzyme by reducing K in the growth medium caused only a small increase in FECa (Fig. 8C).



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Fig. 8. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on FECa in the SER (store Ca): dose response to ouabain presence in the growth medium for 4 days (A), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B), and effect of lowering of medium K for 4 days (C) are shown. A: *significantly higher than the other conditions. B: *significantly higher than Con; {dagger}significantly higher than 1 day ouabain. C: *significantly higher than 5.0 mM and 0.5 mM KCl.

 
Na-K-ATPase inhibition and SOCE. Platelets demonstrate robust SOCE, which largely reflects the status of FECa in the dense tubules (13). A higher FECa is associated with a lower SOCE. Ouabain inhibition of Na-K-ATPase in the CHRF-288 cell caused a dose-responsive decline in SOCE (Fig. 9A). However, there was no apparent effect of K reduction in the growth medium on SOCE (Fig. 9C).



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Fig. 9. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on SOCE: dose response to ouabain presence in the growth medium for 4 days (A), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B), and effect of lowering of medium K for 4 days (C) are shown. A: *significantly lower than the other conditions. B: *significantly lower than Con.

 
Na-K-ATPase inhibition and NCX activity. Ouabain treatment caused a dose-responsive increase in NCX activity (Fig. 10A). Growth medium with lower K concentrations also caused an increase in NCX activity, but this increase was not as pronounced as that with ouabain treatment (Fig. 10C).



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Fig. 10. Effect of Na pump inhibition by ouabain and lowering of K in the growth medium on NCX activity: dose response to ouabain presence in the growth medium for 4 days (A), effect of a fixed ouabain concentration (25 nmol/l) for 1–4 days (B), and effect of lowering of medium K for 4 days (C) are shown. A: *significantly higher than Con; {dagger}significantly higher than the other conditions. B: *significantly higher than Con; {dagger}significantly higher than 1-day ouabain. C: *significantly higher than 5.0 mM KCl.

 

    DISCUSSION
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The central finding of this work is that cultured human megakaryocytes exhibit a coordinated adaptive response to the inhibition of the Na pump. This response includes increased NCX activity, which is consistent with upregulation of the cardiac NCX in rat cardiac myocytes treated with ouabain (18). In addition, inhibition of the Na pump in CHRF-288 cells induced upregulation of SERCA. Apparently, the coordinated increased activity of the NCX and upregulation of SERCA (and perhaps other transport systems not examined in this work) in the CHRF-288 cells served to attenuate the impact of Na pump inhibition on the transmembrane Na and K gradients, which brought about the reduction in Ca extrusion through the NCX. The trade-off for the upregulation of SERCA was an increase in Ca stored in the SER, which led, in turn, to a reduction of SOCE. In previous work (13) using human platelets, we found that acute inhibition of the Na pump also caused an increase in FECa in the dense tubules in concert with a decline in SOCE.

There was a difference in the response of the CHRF-288 cells to inhibition of the Na pump by ouabain vs. K reduction in the growth medium. This difference may relate to the unique characteristics of the megakaryocyte/platelet NCX, which is driven by not only the Na but also the K gradient across the plasma membrane. The Km for K for the exchanger is ~1 mmol/l (12). Therefore, reducing growth medium K not only inhibited the Na pump but also might have caused a lesser drop in the forward mode of the exchanger compared with ouabain treatment. However, this might affect the exchanger activity minimally. Thus other undefined factors may account for a difference in CHRF-288 cell intake with ouabain and K depletion in the medium.

The findings of this in vitro study may have substantial implications for the behavior of human platelets in vivo, given that platelets possess both the {alpha}1-isoform (with low affinity to ouabain) and the {alpha}3-isoform (with high affinity to ouabain) of the Na pump (10). A controversy exists whether in mammals, including humans, endogenous ouabainlike factors circulate at sufficiently high levels to exert biological effects (3, 8, 19). However, recent observations indicate that marinobufagenin, which has high affinity to the {alpha}1-isoform of the Na pump, is an important factor in Na homeostasis in health and disease in mammals, including humans (4–6). Thus, regardless of the controversy about the biological relevance of ouabainlike factors, the presence of both the {alpha}1- and {alpha}3-isoforms of the Na pump in platelets would render platelets responsive to biological agents with the ability to inhibit the Na pump through binding to these subunits. It follows that fluctuating platelet activity might in large measure mirror changing levels of Na pump inhibitors in the circulation.

Nucleated cells that express NCX probably attenuate the impact of Na pump inhibition by the prompt upregulation (and perhaps downregulation) of modalities involved in cellular Na/K/Ca homeostasis, but platelets are anucleated. In CHRF-288 cells, adaptation to Na pump inhibition was mediated by the upregulation of SERCA and increased NCX activity. Such adaptations may not be totally effective in restoring cellular Ca homeostasis to its status before Na pump inhibition, but without them the cells would have experienced a much worse increase in Ca load. Moreover, the experimental circumstances we used to inhibit the Na pump in vitro were considerably harsher than those occurring in vivo, so that changes in cellular Ca status in nucleated cells in the body would not be as pronounced.

The biological life of circulating platelets is roughly 10 days. Thus the effect of persistent increase in levels of Na pump inhibitors on platelet Ca load would ultimately be attenuated, as crops of newly formed platelets are released from megakaryocytes exposed to higher levels of these factors. However, most circulating factors engaged in systemic Na/K regulation, including Na pump inhibitors, express fluctuating levels in response to minute-to-minute or hour-to-hour changes in the overall body load of Na/K. For instance, the episodic ingestion of salt, primarily with meals, generates an array of physiological responses that may acutely increase the circulating levels of Na pump inhibitors. Other circumstances may evoke an acute decline in such factors. Circulating platelets may hence exhibit higher sensitivity to the fluctuating levels of Na pump inhibitors than nucleated cells, which would be ultimately mirrored by acute fluctuations in platelet Ca load and consequently platelet activity. The dependence of the platelet NCX on both the Na and K gradients would further heighten the platelet sensitivity, given that the inhibition of the Na pump not only increases Nac but also diminishes Kc.

Finally, the link between platelet Ca regulation and systemic Na/K homeostasis might explain why occlusive stroke is negatively related to the dietary intake of K (1, 7, 9, 11, 22) and perhaps positively related to the dietary intake of Na (2) by mechanisms that are not always blood pressure mediated. Further support for this concept arises from a recent observation that dietary K supplementation for 3 days had no effect on blood pressure but considerably diminished platelet reactivity (15).


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This work was supported by National Heart, Lung, and Blood Institute Grant HL-63351 and a grant from the Healthcare Foundation of New Jersey.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Aviv, Hypertension Research Ctr., Rm. F-464, MSB, Univ. of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103 (e-mail: avivab{at}umdnj.edu)

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.


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