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
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ABSTRACT |
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platelets; thrombosis; potassium; stroke
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 -subunit isoforms of the Na pump (Na-K-ATPase) in megakaryocytes and 2) the effect of Na-K-ATPase inhibition for 14 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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METHODS |
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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) 0115 Na gluconate, 0115 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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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 (1015 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 -isoforms of the Na-K-ATPase:
1 (catalog no. 05-585) and
2 (catalog no. 06-168) from Upstate Biotechnology (Lake Placid, NY) and
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: 1, 5'-GGCAGTGTTTCAGGCTAA-3' and 5'-TTCATCTGGCAGAAAGAGG-3' (expected product length = 400 bp);
2, 5'-TGGAGACCCGCAATATCTGT-3' and 5'-GTGTTCAATCTCCATTGCT-3' (expected product length = bp);
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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RESULTS |
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DISCUSSION |
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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 1-isoform (with low affinity to ouabain) and the
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
1-isoform of the Na pump, is an important factor in Na homeostasis in health and disease in mammals, including humans (46). Thus, regardless of the controversy about the biological relevance of ouabainlike factors, the presence of both the
1- and
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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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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