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Научни трудове на Съюза на учените в България-Пловдив Серия Г. Медицина, фармация и дентална медицина т.ХХ1. ISSN 1311-9427 (Print), ISSN 2534-9392 (On-line). 2017. Scientific works of the Union of Scientists in Bulgaria-Plovdiv, series G. Medicine, Pharmacy and Dental medicine, VoLXXI. ISSN 1311-9427 (Print), ISSN 2534-9392 (On-line). 2017.
МОДУЛИРАНЕ ФОСФНРТЛТААНЕТН НА Сер-16 В al-ССУБЕДИННЦАТА НА Na/К-ФТФаза ПАТ ЧНВЕШКТ ТРОМБОЦИТИ
Красимир Боянов, Ана Манева
Катедра „Химия и биохимия", Фармацевтичен ^^^i^j^tct, Медицински университет - Пловдив, бул. Васил Априлов15А , Пловдив 4002, България
MODULATIONOF Ser-16 PHOSPHORYLATION IN al-SUBlNiIT OF Na/K-ATPase IN HUMAN PLATELETS Krasimir Boyanov, Ana Maneva DepartmentofChemistry and Biochemistry, F^t^ul^t^f Pharmacy, Medical University - Plovdiv, 15A Vasil AprilovBlvd., 4002 Plovdiv, Bulgaria
Abstract
Na/K-ATPase is a membrane-bound enzyme lhal generates an electrochemical gradient ariliaal to the regulation of cell volume, intracellular pH and Ca2+-levels in platelets. The activity of the enzyme is regulated by phosphorylation of serine-16 in the a1-subunit of Na/K-pump: increased phospho-Ser-16, reduces pump activity. Wortmannin, caffeine, quercetin (inhibitors of platelet aggregation) and ouabain (activator of platelets) inhibit Na/K-ATPase activity and/or increase phosphorylation of Ser-16 in different cell types. Lactoferrin is a natural anticoagulant that stimulates the activity of Na/K-pump in erythrocytes. In the present study we examine the effect of lactoferrin, wortmannin, caffeine, quercetin and ouabain on the degree of phosphorylation of Ser-16 in human platelets, using cell-based ELISA. The results obtained indicate that lactoferrin itself, does not affect phospho-Ser-16, while wortmannin, caffeine, quercetin and ouabain, applied alone and in combination with lactoferrin, enhance Ser-16 phosphorylation in the a1-subunit of Na/K-ATPase.
Keywords: Phosphorylation, Ser-16, a1-subunit, Na/K-ATPase, Platelets INTRODUCTION
Na/K-ATPase, also known as sodium pump, is a membrane-bound enzyme that uses the energy from the hydrolysis of one molecule of ATP, to transports three Na+ out in exchange for two K+ that are taken in.
Structurally, the Na/K-ATPase is an oligomer that is composed of distinct molecular forms of two major polypeptides, the a and в subunits (Blanco, 2005). The a-subunit is a membrane protein which repeatedly crosses the plasma membrane; it is also responsible for the catalytic and transport properties of the enzyme and it contains binding sites for cations, ATP, and the inhibitor, ouabain (Schwinger et al., 2003; Morth et al., 2009). The в-subunit is a polypeptide that crosses the membrane once; it is essential for the normal activity of the enzyme and it modulates the affinity of the sodium pump to Na+ и K+-ions (Geering, 2001). At present, four structural variants of the a (a1, a2, a3 and a4) polypeptide (Lingrel et al., 2007) and three в (в1
f2 and f3) subunits (Geering, 2001) have been identified in mammals. A third protein, termed the y-subunit, has also been identified (Tejral et al., 2017). Association of the a and f polypeptides in different oligomers results in multiple isozymes of the Na/K-ATPase that have unique functional properties and a tissue-specific pattern of expression (Blanco, 2005). The al and f1 isoforms constitute the Na/K-ATPase isozyme that is expressed ubiquitously in tissues (Tokhtaeva et al., 2012). Platelets have only fl-isoform of f subunit (Stengelin & Hoffman, 1997). The electrochemical gradient the Na/K-ATPase generates is critical in maintaining cell volume, intracellular pH and Ca2+-levels in platelets. (Borin & Siffert, 1991; Aviv, 1992; Marx et al., 1992; Rosskopf, 1999).
Although it is known that the sodium pump activity is regulated by the intracellular and extracellular ionic composition, regulation can also occur through phosphorylation of the a subunit by various kinases (Therien & Blostein, 2000). Feraille et al. (2000) discovered that there are complex regulatory mechanisms that affect the activity of Na/K-ATPase after stimulation of phorbol ester-sensitive protein kinase C (PKC) - incubation with 10-7 M phorbol 12,13-dibutyrate for 15 min at 37°C, inhibits the activity of sodium pump in COS-7 cells, which is due to phosphorylation of Serine-16 (Ser-16) in a1 subunit by the activated PKC. So far it has not been investigated in human platelets whether the degree of phosphorylation of Ser-16 in the catalytic a1 subunit of Na/K-ATPase is affected.
The purpose of the present study is to investigate the effects of lactoferrin, wortmannin, caffeine, quercetin (platelet aggregation inhibitors) and ouabain (platelet activator) on the degree of phosphorylation of Ser-16 in a1-subunit of Na/K-pump in human platelets. It was studied whether these modulators, with proven effects on coagulation, may influence the intracellular signaling, mediated by the processes of phosphorylation in these cells.
MATERIALS AND METHODS
Isolation of platelets
Platelets were obtained from human blood of 10 healthy donors using sodium citrate. Platelet-rich plasma was separated by centrifugation at 400 g for 10 min, followed by centrifugation for 15 min at 1100 g to obtain platelets (Maneva et al., 1993). After being washed three times with 1 mM EDTA/phosphate-buffered saline (PBS), pH 7.4, and once with PBS, pH 7.4, platelets were suspended in 50 mM PBS (Maneva et al., 1993) to a final concentration of 1.5 x 105 cells/probe. All the procedures described were carried out at room temperature. Determination of Ser-16 phosphorylation
A cell-based colorimetric ELISA was used for in vitro determination of the phosphorylation level of Ser-16 in Na/K-ATPase. The experiment was performed with "Phospho-ATP1A1 (Ser16) Colorimetric Cell-Based ELISA Kit" (Aviva Systems Biology, USA). In accordance with the protocol, two types of primary antibodies were used - the first type is specific for the a-subunit of Na/K-ATPase phosphorylated at Ser-16 (Anti ATP1A1 P-Ser 16 - A) and the second type is specific for the a-subunit of Na/K-ATPase (Anti ATP1A1 - B).
Following the protocol, first, we placed the isolated platelets (1.5 x 105 cells/well) in each of the wells (Versteeg et al., 2000), and incubated the plate overnight at 37°C. Then, the platelets were treated with modulators separately and in combination with lactoferrin for 30 min at 37°C (Cesar et al., 2006). The agents were applied at the following concentrations: lactoferrin (25 nM), wortmannin (50 nM), caffeine (20 mM), quercetin (1.5 ^M), ouabain (0.5 mM), lactoferrin (25 nM) + wortmannin (50 nM), lactoferrin (25 nM) + caffeine (20 mM), lactoferrin (25 nM) + quercetin (1.5 ^M), lactoferrin (25 nM) + ouabain (0.5 mM). The untreated cells were regarded as control, while the cells to which the modulators were administered were regarded as samples. Both the samples and control were incubated under the same conditions. After incubation, the platelets were washed twice with TBS and fixed with 8 % formaldehyde. Several washing steps and addition of blocking buffer preceded the addition of primary antibodies (each of them in a single well). Then, secondary antibodies conjugated with horseradish peroxidase were applied, and
finally, the substrate for this enzyme. The absorbance was measured after the addition of stop solution at 450 nm wavelength against blank.
The OD (optical density) values obtained for the phosphorylated target protein were normalized using the OD values obtained for the non-phosphorylated target protein via the proportion, OD450 (Anti-ATP1A1 P-Ser16 Antibody)/OD450 (Anti-ATP1A1 Antibody) - A/B. Statistical analysis
Statistical analyses were performed using independent T-test. The results are expressed as means ± standard deviation. Values were considered statistically significant when the p-value was less than 0.05. The effect of modulators is represented as percentages.
RESULTS
Table 1 shows the mean absorption values obtained by incubating with the two different primary antibodies (A and B) after treating the platelets with the modulators, the proportions -A/B and the level of significance (p) showing how statistically significant is the effect on the phosphorylation of Ser-16 under the action of each one of the modulators. The mean absorption values were obtained by ten-time repetitions (n=10).
Table 1. Phosphorylation of Ser-16 in a!-subunit of Na/K-ATPase.
Antibodies Anti ATP1A1 P-Ser 16 - A (Xod450 ± SD) Anti ATP1A1 - B (Xod450 ± SD) p A/B
Without an agent 0.146 ± 0.035 1.018 ± 0.340 < 0.001 0.143
Lactoferrin (25 nM) 0.127 ± 0.037 0.896 ± 0.371 < 0.001 0.142
Wortmannin (50 nM) 0.134 ± 0.030 0.849 ± 0.258 < 0.001 0.158
Caffeine (20 mM) 0.140 ± 0.036 0.893 ± 0.318 < 0.001 0.157
Quercetin (1.5 ^M) 0.145 ± 0.034 0.929 ± 0.207 < 0.001 0.156
Ouabain (0.5 mM) 0.145 ± 0.030 0.886 ± 0.178 < 0.001 0.164
Lactoferrin (25 nM) + Wortmannin (50 nM) 0.145 ± 0.023 0.864 ± 0.179 < 0.001 0.168
Lactoferrin (25 nM) + Caffeine (20 mM) 0.158 ± 0.037 0.843 ± 0.182 < 0.001 0.187
Lactoferrin (25 nM) + Quercetin (1.5 ^M) 0.155 ± 0.016 0.861 ± 0.038 < 0.001 0.180
Lactoferrin (25 nM) + Ouabain (0.5 mM) 0.167 ± 0.022 0.848 ± 0.172 < 0.001 0.197
Legend: Xod450 - mean absorption value obtained at 450 nm wavelength; SD - standard deviation; p - level of significance; n - number of repetitions.
All the modulators used, alone and in combinations, at the concentrations administered, influenced the phosphorylation of Ser-16 significantly (Table 1).
To find the effect of each of the modulators on phospho-Ser-16, according to the manufacturer, the proportions in Table 1 (Anti ATP1A1 P-Ser 16/Anti ATP1A1 = A/B) for the samples and for the control are juxtaposed with one another, for example, A/BLactofemn (25 nM)/A/Bwithout an agent, A/Bwortmanmn (50 nM)/A/Bwithout an agent, etc. The values obtained show the level of increase or decrease of phosphorylation in the samples, compared to the control. The values of increase or decrease are presented in Figure 1 in percentages, taking 100 % to be the phosphorylation of Ser-16 in the control - cells not treated with an agent.
Wortmannin (50 nM), caffeine (20 mM), quercetin (1.5 pM) and ouabain (0.5 mM) stimulate Ser-16 phosphorylation in a1-subunit of Na/K-ATPase from 9.09 % to 14.69 % (Figure 1A). Lactoferrin (25 nM) increases the stimulating effect of the four modulators, as administered together with them it enhances additionally the phosphorylation of Ser-16 - from 17.48 % to 37.76 % (Figure 1B). However, lactoferrin itself did not affect phosphorylation of the serine residue (Figure 1).
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Figure 1. Phosphorylation of Ser-16 (%) under the action of modulators alone (A) and in combination with lactoferrin (B).
Phosphorylation of Ser-16 in control (cells not treated with an agent) is taken as 100 %.
Lf - lactoferrin, Wort - wortmannin, Caff - caffeine, Querc - quercetin, Ouab - ouabain.
Without 25 nM Lf 25 nM Lf 25 nM Lf 25 nM Lf 25 nM Lf an agent + 50 nM + 20 mM + 1.5 jM + 0.5 mM
Wort Caff Querc Ouab
DISCUSSION
Platelets are targets of Na/K-ATPase inhibitors - specific ouabain-binding sites have been found on their surface (Bork & Mrsny, 1993). It is well-known that ouabain inhibits the Na-pump in human erythrocytes and in T84 cells (Whittam et al., 1962; Ecay et al., 2000), as well as it increased Ser-16 phosphorylation in a1-subunit of sodium pump in rat parotid acinar cells (Soltoff et al., 2010). Our findings showed that ouabain elevates Ser-16 phosphorylation in a1-subunit of Na/K-ATPase in human platelets by 14.69 % (Figure 1A), which suggests a reduced membrane pump activity, and this coincides with the observation that it forms a complex with the a1-subunit in these cells (Blanco & Mercer, 1998).
Through in vitro experiments, Ogundajo et al. (2014) found that quercetin is an inhibitor of the brain Na/K-ATPase in rats. A year later, Ogundajo and Imoru (2015) found, through in vitro and in vivo experiments, that quercetin also inhibits the liver Na/K-ATPase in rats. These data support our results according to which quercetin increases the level of phosphorylation of Ser-16 in human platelets by 9.09 % (Figure 1A), and this suggests a reduced activity of the membrane
enzyme (Féraille et al., 2000). Probably, increased phosphorylation of Ser-16 in the a1-subunit of the pump (Figure 1A) is one of the mechanisms through which quercetin inhibits the activity of Na/K-ATPase.
Wortmannin inhibits Na/K-ATPase in human colonic cell line T84 (Ecay et al., 2000). Our findings showed that wortmannin increases Ser-16 phosphorylation of Na-pump in human platelets by 10.49 %, in comparison with the control (Figure 1A). This means that the modulator used influence the phosphorylation of this key amino acid residue as well, and hence the Na/K-ATPase activity, as according to the literature data, it is reduced (Féraille et al., 2000). Since wortmannin is a specific inhibitor of phosphoinositide 3-kinase - PI3K (Yano et al., 1993), the data from the study show that PI3K-dependent pathway is included in the control of Na/K-ATPase (Figure 1A).
Caffeine is an inhibitor of phosphodiesterases (Feneck, 2007). It also inhibits Na/K-ATPase activity by 62 % in rat pancreatic islets (Tung et al., 1990). In 2002, Lee et al. proved that in kidney cells of caffeine-treated rats the expression of a1 and ^1-subunits of Na/K-pump decreases, as well as that of Na+/H+-exchanger 3 (NHE3), which in turns, leads to a decrease of the catalytic activity of Na/K-ATPase, too. It has not been found any caffeine influence on the human platelet sodium pump, as well as how this methylxanthine affects Ser-16 phosphorylation in the a1-subunit. Our findings showed that caffeine increases the level of Ser-16 phosphorylation by 9.79 %, in comparison with the control (Figure 1A), which means that the phosphodiesterase inhibitor influences the phosphorylation of this key amino acid residue as well, and hence the Na/K-ATPase activity, as it is reduced (Féraille et al., 2000). The results obtained show a probable involvement of a cAMP-dependent pathway (Lev et al., 2007) and/or of caffeine-sensitive protein kinases (Biovin et al., 1988).
Lactoferrin does not affect Ser-16 phosphorylation in human platelets, as it decreases by only 0.7 % (Figure 1). However, lactoferrin increases the tyrosine phosphorylation of membrane proteins (Kobayashi et al., 2005). In renal epithelial cells, it has found that a1-subunit of Na/K-ATPase may be phosphorylated on Tyrosine-537 by tyrosine kinases, and this phosphorylation is necessary for initiating an intracellular signaling pathway (Doné at al., 2002). Lactoferrin activates Na/K-ATPase in erythrocytes and this might be due to either its stimulating effect on NHE (Sun et al., 1991) or to some changes in enzyme phosphorylation (Maneva et al., 2007). The presence of lactoferrin receptors on the platelet membrane surface has been established (Maneva et al., 1993). Lactoferrin-receptor interaction may cause conformational changes altering the accessibility to enzymes phosphorylating Na-pump: protein kinase A (PKA) and PKC may phosphorylate a-subunit in a certain conformation, depending on the cell type (Feschenko & Sweadner, 1994).
On the other hand, lactoferrin increases the stimulatory effects of wortmannin, caffeine, quercetin and ouabain on Ser-16 phosphorylation (Figure 1B). This shows the involvement of PI3K (wortmannin), cAMP-PKA-pathway (caffeine), changes in membrane redox state (quercetin), changes in the level of phosphorylation of proteins (wortmannin, caffeine, quercetin and ouabain) in the mechanisms of lactoferrin biological activity in platelets.
In our experiments, wortmannin, caffeine, quercetin and ouabain stimulate phosphorylation of Ser-16 (Figure 1A). The increased phosphorylation of this amino acid residue is related to the reduced activity of the Na/K-ATPase (Féraille et al., 2000). Some authors have observed sodium pump endocytosis (Ecay et al., 2000; Khundmiri et al., 2004) that requires ERK-dependent phosphorylation of Ser-16 in a1-subunit of the membrane enzyme (Khundmiri et al., 2004). According to Ecay et al. (2000), such endocytosis decreases the overall activity of the Na/K-ATPase, but it does not reduce the total content of the enzyme in the cell, which means that it does not interrupt the signal pathway that originates from the pump. In cardiac myocytes, the binding of ouabain to Na/K-ATPase initiates a signalosome formation on the membrane, involving a large number of molecules from which an intracellular signaling pathway that stimulates cell growth begins (Xie & Cai, 2003). In a similar mechanism, wortmannin, caffeine and quercetin can also initiate changes in cellular signaling in human platelets, which may affect the behavior of these cells.
This indicates that the agents used can be both modulators of platelet aggregation and of
intracellular signaling pathways in human platelets.
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