Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap4A (diadenosine tetraphosphate), Up4A (uridine adenosine tetraphosphate) and Ap5A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [3H]AMP and [3H]ADP into [3H]ADP/ATP and [3H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [3H]ADP and [3H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC50 values (−log IC50): Ap4A, 4.67±0.03; Up4A, 3.70±0.10; Ap5A, 6.31±0.03; suramin, 3.74±0.07], as well as on endothelial adenylate kinase (pIC50 values: Ap4A, 4.17±0.07; Up4A, 2.94±0.02; Ap5A, 5.97±0.04; suramin, 4.23±0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap4A, Up4A, Ap5A and suramin progressively inhibited the rates of [3H]ADP (pIC50 values: Ap4A, 3.38±0.09; Up4A, 2.78±0.06; Ap5A, 4.42±0.11; suramin, 4.10±0.07) and [3H]ATP (pIC50 values: Ap4A, 3.06±0.06; Ap5A, 3.05±0.12; suramin, 4.14±0.05) hydrolyses by cultured HUVECs. Up4A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap4A, Up4A and Ap5A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 μmol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap4A and Ap5A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up4A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.
The potent actions of mononucleoside polyphosphates in the cardiovascular system were first described in 1929 . It is now well known that mononucleoside polyphosphates are involved in various vasoregulatory processes, and immunomodulatory and prothrombotic responses in humans [2,3]. The physiological effects of mononucleoside polyphosphates are mediated by the nucleotide-selective receptors P2X and P2Y . P2X receptors are ligand-gated ion channels, which are opened by purinergic messengers . Hereby, rapid changes in the membrane permeability of monovalent and divalent cations are mediated [6,7]. P2Y receptors are seven-transmembrane-spanning proteins and belong to the superfamily of G-protein-coupled receptors [4,8]. P2Y receptors act by a downstream signalling cascade, including G-proteins and inositol triphosphate among other factors [4,9], through the activation of phospholipase C and/or regulation of adenylate cyclase activity [10,11].
In recent years, dinucleoside polyphosphates gained increasing interest as another group of potent P2X and P2Y receptors agonists. Dinucleoside polyphosphates contain two purine or pyrimidine bases, which are interconnected by a phosphate chain with a variable number of phosphates. Firstly, diadenosine polyphosphates (ApnA, n=2–7) were isolated from body fluids and cells (e.g. [12–16]). Diadenosine polyphosphates may serve as important neurotransmitter molecules in the nervous system  and stimulate different responses in the cardiovascular system, controlling vascular tone and preventing platelet aggregation [15,18]. For example, in mammalian cells, the intracellular concentration of Ap4A (diadenosine tetraphosphate) during normal growth  correlates directly with the proliferative state of the cell or tissue [20,21]. Furthermore, Ap4A and Ap5A (diadenosine pentaphosphate) are characterized as having strong vasoregulatory properties (e.g. ), and the corresponding plasma concentrations are sufficient for vasoregulatory effects .
Another dinucleoside polyphosphate, Up4A (uridine adenosine tetraphosphate), was isolated from the supernatant of stimulated human endothelium . The potent vasoconstrictive effects of Up4A and its release upon endothelial stimulation strongly suggest that Up4A has a functional vasoregulatory role. In healthy subjects, plasma concentrations of Up4A are found to be sufficient to cause vasoconstriction , and its concentration is significantly increased in hypertensive subjects and correlates with the intima-media thickness, as well as with the left ventricular mass of these patients caused by the proliferative effects of Up4A .
Formerly, inactivation of the released nucleoside polyphosphates was thought to be mainly regulated by vascular endothelial  and lymphoid  membrane-bound NTPDase (nucleoside triphosphate diphosphohydrolase; also known as ecto-ATPDase, CD39) and ecto-5′-nucleotidase (CD73). Ecto-hydrolases are present on a broad variety of cell types, including aortic endothelial cells [27,28], chromaffin cells , rat mesangial, bovine corneal epithelial, human hepatoma cells (Hep-G2) and peridontal cells .
In contrast with these traditional paradigms, it has now become clear that the nucleotide-phosphorylating enzymes adenylate kinase and NDP kinase are also co-expressed on the cell surface and finely control the purinergic signalling cascade via two counterbalancing, nucleoside-polyphosphates-inactivating and nucleoside-polyphosphates-regenerating, pathways [31,32]. The identification of a complex mixture of NPP (nucleotide pyrophosphatase/phosphodiesterase), NTPDase, adenylate kinase and other soluble purinergic enzymes freely circulating in the bloodstream adds another level of complexity to the understanding of the regulatory mechanisms of purine homoeostasis within the vasculature [33–35].
Therefore, in the present study, we investigated the effects of Ap4A, Ap5A (diadenosine pentaphosphate) and Up4A on human serum and vascular endothelial adenylate kinase and other nucleotide-converting activities. These comparative competitive results suggest that Ap4A and Ap5A could contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up4A most probably mediates its vasoregulatory effects via direct binding-mediated mechanisms.
MATERIAL AND METHODS
Synthesis of Up4A
Up4A is not commercially available, therefore this dinucleoside polyphosphate was synthesized as described by Jankowski et al. . Briefly, ATP (0.25 mmol/l), uridine 5′-monophosphate (0.25 mmol/l), Hepes (2 mol/l), N-ethyl-N′-(3-dimethylaminopropyl)carbodi-imide (2.5 mol/l) and MgCl2 (125 mmol/l) were incubated at 37°C for 24 h. The mixture was fractionated to homogeneity (<98%) by reverse-phase and anion-exchange chromatographies . The resulting fractions were freeze-dried and examined by MALDI (matrix-assisted laser-desorption ionization) MS. All other nucleotides and dinucleotides were obtained from Sigma–Aldrich.
Measurement of serum nucleotide-converting activities
For serum preparation, 10 ml of blood was taken from the antecubital vein of four healthy females (31±4 years) after giving their written consent to participate in the study. Blood samples were allowed to clot at room temperature for 20 min before centrifugation (10 min at 2000 g, 6°C). The studies were approved by the Ethics Committee of Turku University. Supernatants were additionally centrifuged for 10 min at 15000 g, followed by freezing the resultant serum at −40°C. In the case of inhibitory studies, serum was pre-incubated at 37°C for 20 min without (control) and with various concentrations of Ap4A, Up4A, Ap5A and suramin as potential inhibitors. Purinergic activities were then assayed by incubating the treated serum at 37°C in a final volume of 80 μl of RPMI 1640 medium containing 5 mmol/l β-glycerophosphate, unlabelled nucleotides and tracer [2,8-3H]ADP (PerkinElmer, Boston, MA, U.S.A.) and [2-3H]AMP (Amersham, Little Chalfont, Bucks., U.K.) as appropriate substrates. Specifically, for adenylate kinase and NDP kinase activities, serum (5 μl) was incubated with 500 μmol/l [3H]AMP or [3H]ADP as respective phosphate acceptors and 800 μmol/l of γ-phosphate-donating ATP. Incubation times varied from 30 to 50 min, so that the amount of converted nucleotides did not exceed 8–15% of the initially introduced substrate. Samples were applied on to Alugram SIL G/UV254 sheets (Macherey-Nagel, Dueren, Germany). Radiolabelled substrates and their products were separated by TLC and the radioactivity quantified by scintillation β-counting .
Measurement of endothelial nucleotide-converting activities
HUVECs (human umbilical vein endothelial cells) (passages 2–3) were seeded on to gelatin-coated 96-well tissue culture plates (Greiner-Bioone, Frickenhausen, Germany) at a density of 15000 cells/well in complete medium, as described previously . Later (24 h), HUVECs were rinsed with RPMI 1640 medium and pre-incubated with various inhibitors for 20 min prior to the addition of nucleotide substrates. Ecto-nucleotidase activities were measured by incubating HUVECs for 40 min at 37°C in 200 μl of RPMI 1640 medium containing 5 mmol/l β-glycerophosphate and 400 μmol/l [3H]ADP or [2,8-3H]ATP (PerkinElmer) as preferred substrates for NTPDase. For adenylate kinase activity, HUVECs were incubated for 40 min with 400 μmol/l [3H]AMP in the presence of 800 μmol/l unlabelled ATP. Applying the samples of the mixtures on to the TLC sheets, as described above, terminated the enzymatic reaction.
Determination of the half-life of Ap4A, Up4A and Ap5A in human serum
In order to determine the half-life in human serum, Ap4A, Up4A or Ap5A (100 μg of each) was added to 12 ml of human serum. The mixture was incubated at 37°C; aliquots (2 ml) were collected after 0, 2, 4, 10, 60 and 120 min. Ap6A (diadenosine hexaphosphate) (40 μg) was added to the samples as an internal standard, as the chromatographic characteristics, as well as the degradation kinetics, of Ap6A are comparable with Ap4A, Up4A and Ap5A. The samples were deproteinized with 0.6 mol/l (final concentration) perchloric acid, and the precipitated proteins were removed by centrifugation (3000 g, 4°C, 10 min). After adjusting the pH to 9.0 with 5 mol/l KOH, KClO4 was removed by centrifugation (3000 g, 4°C, 5 min).
TEAA (triethylammonium acetate) in water was added to the deproteinized plasma to give a final concentration of 40 mmol/l. This mixture was loaded on to a preparative reverse-phase HPLC column (Chromolith Performance, RP-18 e, 100 mm×4.6 mm; Merck, Darmstadt, Germany) (equilibration and sample buffer, 40 mmol/l TEAA in water; flow rate, 1 ml/min). Ap4A, Up4A, and Ap5A were eluted with 24% acetonitrile in water and freeze-dried.
The freeze-dried fractions of the reversed-phase chromatography were analysed by MALDI MS. The freeze-dried fractions were resuspended in 10 μl of water. The analyte solution (1 μl) was mixed with 1 μl of matrix solution (50 mg/ml 3-hydroxy-picolinic acid, in water). To this mixture, cation exchange beads (AG 50 W-X12, 200–400 mesh; Bio-Rad, Munich, Germany) were added, and equilibrated with NH4+ as a counter-ion to remove Na+ and K+ ions. The mixture was dried gently on a prestructured MALDI sample support (MTP AnchorChip™ 400/384, Bruker Daltonics, Bremen, Germany) before introducing it into the mass spectrometer. The nucleotide amount was calculated by the mass intensity, as described previously . The mass accuracy was in the range of 0.01%. Local differences in the nucleotide concentration of the MALDI spot were neutralized by the use of internal standard.
Data from competitive experiments were subjected to computer analysis by non-linear least-squares curve fitting to determine log IC50 values (GraphPad Prism™, version 5.01; GraphPad, San Diego, CA, U.S.A.). The results are presented as the means±S.E.M. Results were tested for statistical significance using ANOVA for repeated measurements. The constants are expressed in terms of pIC50 (−log IC50)±S.E.M. for at least three independent experiments. All results are given as means±S.E.M. for the individual values indicated.
As human blood contains an extensive network of soluble purinergic enzymes , we evaluated the effects of dinucleoside polyphosphates, as well as the non-specific P2-receptor antagonist suramin, on the pattern of nucleotide metabolism. Firstly, serum adenylate kinase activity was determined by TLC as the rate of [3H]AMP transphosphorylation with unlabelled ATP into high-energy 3H-labelled phosphoryls. Human serum caused specific ATP-mediated phosphorylation of [3H]AMP, and this soluble adenylate kinase reaction was progressively inhibited in the presence of increasing concentrations of dinucleoside polyphosphates and suramin (Figure 1A). The pIC50 values for this inhibitory effect were 4.67±0.03, 3.70±0.10, 6.31±0.03 and 3.78±0.07 for Ap4A, Up4A, Ap5A and suramin respectively. These values are within the physiological range for diadenosine polyphosphates released by adrenal glands into plasma . Since Up4A is released from endothelial cells after stimulation , local Up4A concentrations are significantly increased compared with the Up4A plasma concentration. Therefore, the effects of Up4A on the soluble adenylate kinase are of great importance for cardiovascular regulation, although the overall plasma concentration of Up4A is below the IC50 value. In principle, human blood contains two soluble enzymes, adenylate kinase and NTPDase, both of which can use ADP as substrate [34,35]. Pre-incubation of human serum with Ap5A prior to addition of [3H]ADP was accompanied by marked (approx. 80%) decrease in the rate of [3H]ADP conversion into [3H]AMP, indicating that adenylate kinase represents the major ADP-converting enzyme in cell-free human serum. The remaining NTPDase activity (determined in the presence of excess Ap5A) was then taken into account as a basal value during subsequent quantification of adenylate kinase activity. Dinucleoside polyphosphates do not only have an inhibitory effect on soluble adenylate kinase, but also an inhibitory effect on membrane-bound endothelial adenylate kinase (Figure 1B). The pIC50 values for this inhibitory effect were 4.17±0.07, 2.94±0.02, 5.97±0.04 and 4.22±0.07 for Ap4A Up4A, Ap5A and suramin respectively (Figure 1B).
Effects of Ap4A, Up4A, Ap5A and suramin on adenylate kinase activity in human serum (A) and cultured endothelial cells (B)
Next, the activity of the soluble nucleotide-phosphorylating enzyme NDP kinase was quantified by using [3H]ADP and ATP as the γ-phosphate acceptor and donor respectively. Serum pre-treatment with suramin was accompanied by progressive inhibition of the rate of ATP-dependent [3H]ADP phosphorylation into [3H]ATP (pIC50=3.86±0.08), whereas Ap4A, Up4A and Ap5A were unable to significantly inhibit serum NDP kinase activity, even at non-physiological concentrations (Figure 2).
Effects of Ap4A, Up4A, Ap5A and suramin on soluble NDP kinase activity in human serum
We also evaluated the effects of these purinergic agents on the rates of [3H]ATP and [3H]ADP hydrolysis. Suramin and dinucleotide polyphosphates only moderately inhibited endothelial ADP-hydrolysing activities (Figure 3A). The pIC50 values for this inhibitory effect were 3.38±0.09, 2.78±0.06, 4.42±0.11 and 4.10±0.07 for Ap4A, Up4A, Ap5A and suramin respectively. Similarly, measurement of endothelial NTPDase activity by using ATP as another preferred substrate revealed that Ap4A (pIC50=3.06±0.06), Ap5A (pIC50=3.05±0.12) and suramin (pIC50=4.14±0.05), but not Up4A, progressively decreased the rate of [3H]ATP hydrolysis by cultured HUVECs (Figure 3B).
Effects of Ap4A, Up4A and suramin on the rates of [3H]ADP (A) and [3H]ATP (B) hydrolyses by cultured HUVECs
These results indicate that, in contrast with suramin, which non-specifically affects most of the studied activities, the inhibitory effects of Ap5A, and to lesser extent of Ap4A, on adenylate kinase activity occur at low micromolar and even sub-micromolar range and cannot be extrapolated on the other nucleotide-converting enzymes, NDP kinase and NTPDase. Next, we estimated the ability of cell-free human serum to degrade Ap4A and Up4A to verify the effects of degradation products of dinucleoside polyphosphates. The half-lives of Ap4A, Up4A and Ap5A in human serum were approx. 9.8±4.6, 4.4±2.1 and 10.4±3.5 min respectively.
Dinucleoside polyphosphates have the capacity to potentiate signalling effects via P2 receptors (primarily, via P2X1, P2X3 and P2Y1 subtypes) [15,23], although the existence of specific dinucleoside polyphosphate receptors have also been proposed [17,18,39,40]. However, the specificity of dinucleotide receptors and their involvement in multiple extracellular signals are still at the beginning of being understood, mostly due to the complexity of purinergic signalling cascades.
Released dinucleoside polyphosphates are hydrolysed by enzymes of the NPP-type ecto-phosphodiesterase family ; therefore, the overall response of cells should be the integral of various effects of dinucleoside polyphosphates and their biologically active metabolites, ATP, ADP or adenosine, as well as uridine analogues. However, although the half-life times for the dinucleoside polyphosphates are comparable with the incubation times, the contribution of these metabolites to the effects observed in the present study are not relevant, since the concentration of metabolites formed are relatively insignificant compared with the 800 μmol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assay.
Dinucleoside polyphosphates potentiate and markedly sensitize the purinergic effects of ATP in cultured cerebellar astrocytes, and these effects may occur via binding of this dinucleotide with its own, dinucleoside-polyphosphate-specific, receptor and the activation of the ERK (extracellular-signal-regulated kinase) signalling cascade . The identification of the membrane-bound and soluble forms of adenylate kinase , and powerful inhibition of this catalytic reaction by Ap5A , could provide another reasonable explanation for agonistic effects of the bi-substrate analogue.
The recent results suggest that, in addition to the strong direct effects, dinucleoside polyphosphates most probably mediate part of their effects in the physiological range via potent inhibition of membrane-bound and soluble adenylate kinases, thus preventing subsequent metabolism of the important purinergic agonist ADP. Indeed, the ability of dinucleoside polyphosphates to stimulate the endocytosis of high-density lipoproteins in human hepatocytes is mediated via inhibition of ecto-adenylate-kinase-mediated conversion of endogenous ADP, with subsequent stimulation of P2Y13 receptors . Similarly, results regarding the ability of dinucleoside polyphosphates to inhibit the pattern of extracellular ATP catabolism  has to be taken into consideration during evaluation of agonistic potency for dinucleoside polyphosphates. Ap4A and Ap5A might also inhibit both adenylate kinase and NTPDase activities, and hereby potentiate their agonistic effects. Since diadenosine polyphosphates are stored and released together with the corresponding mononucleoside polyphosphates, the overall physiological effects, such as thromboregulation, vasodilatation, vasoconstriction and platelet activation, may be proposed to be due to competition between degradation of diadenosine polyphosphates and degradation of mononucleoside polyphosphates.
In contrast, the inability of Up4A to inhibit endothelial and serum purinergic enzymes support the hypothesis that certain receptors mediate the vasoregulatory effects of this dinucleoside polyphosphate, rather than secondary inhibitory effects.
In conclusion, the agonistic effects of dinucleoside polyphosphates are obviously mediated by complex mechanisms: (a) via dinucleoside polyphosphate or mononucleoside polyphosphate receptor pathways, (b) by decreasing the ecto-adenylate-kinase activity and (c) by generation of biologically active mononucleoside polyphosphates and nucleosides in the course of their degradation.
human umbilical vein endothelial cell
matrix-assisted laser-desorption ionization
nucleoside triphosphate diphosphohydrolase
uridine adenosine tetraphosphate
The present study was supported by a grant from the Finnish Academy and Sigrid Juselius Foundation (G.G.Y. and S.J.), the German Research Foundation (DFG, Ja-972 /11-1 to J.J.), Sonnenfeld Foundation (J.J.) and by a Rahel-Hirsch Scholarship from the Charité (V.J.).