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Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, University of Liverpool, L69 3GE, UK; and UK Centre for Tissue Engineering,
* Department of Clinical Engineering, University of Liverpool, L69 3GA, UK
1Correspondence: Department Human Anatomy and Cell Biology, The Sherrington Buildings, Ashton St., University of Liverpool, L69 3GE, UK. E-mail: buckleyk{at}liv.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto-NDPK activity; confirming that the combined action of multiple osteoblast-expressed ecto-enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP-consuming, and for the first time, ATP-generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone.
Key Words: ATP • ADP • bone • ecto-nucleoside diphosphokinase • ecto-NDPK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and this receptor family was subsequently subdivided into G-protein-coupled P2Y receptors and P2X ligand-gated ion channels (2)
. Currently, seven mammalian P2X receptors, P2X1-7, and six P2Y receptors, P2Y1,2,4,6,11,12, have been identified. Diverse responses to extracellular nucleotides have been described in a wide range of tissues and biological systems.
Osteoblasts are bone-forming cells that produce and secrete the bone matrix constituents and regulate bone resorption. These cells express multiple subtypes of P2X and P2Y receptors (3
4
5
6
7
8)
. Nucleotides influence many of the processes that govern skeletal remodeling, the highly coordinated process of bone regeneration where old tissue is replaced by newly synthesized bone. ATP stimulates osteoblast proliferation (9)
, DNA synthesis (6)
, and up-regulates their expression of receptor activator for NF
B ligand (RANKL) (10)
, the essential factor in inducing osteoclastogenesis (11)
. Numerous studies have also reported expression of multiple P2 receptor subtypes by the bone-resorbing osteoclast cells (12
13
14)
; activation of these receptors is thought to increase both osteoclast formation and resorption (10
, 15
16
17
18)
.
Extracellular nucleotides are clearly important to osteoblast and osteoclast function; therefore, the mechanisms that result in the presence of nucleotides such as ATP in the extracellular environment and how they are regulated is of interest. ATP is a ubiquitous intracellular molecule, and consequently every cell is a potential source of this nucleotide via cell lysis. Adenine and uridine nucleotides may be transported into the extracellular environment nonlytically via their inclusion in exocytotic vesicles (19
20
21)
. Numerous cell types have been shown to release ATP constitutively (22
, 23)
, and previous preliminary investigations by our group suggested that this could be observed in osteoblasts (24)
. Mechanical stimulation is thought to increase this release (25
, 26)
, indicating that nucleotides may play a role in mechanotransduction in bone, the process by which detection of mechanical deformation or fluid shear by skeletal cells results in remodeling.
Of equal importance is the life span of nucleotides once outside the cell. Nucleotidase enzymes are present on the extracellular side of osteoblast membranes and are capable of removing phosphate groups from nucleotides (27)
. Therefore, ATP or UTP in the presence of ecto-nucleotidases are rapidly converted to A/UDP, then A/UMP, and finally to adenosine/uridine, thereby terminating the potential activation of P2 receptors (28)
. Recently, however, increasing attention has been given to the alternative possibility of ATP synthesis from ADP. Although nucleoside diphosphokinase enzymes are well established as maintaining the balance of ribo- and deoxyribonucleoside triphosphates inside the cell (29)
and perform a crucial role in the citric acid chain, a number of studies have recently reported the presence of these enzymes on extracellular cell surfaces (30
31
32
33)
. Nucleoside diphosphokinase enzymes, encoded by the human nm23-H1 gene, catalyze the transfer of the terminal phosphate group of 5'-triphosphate nucleotides to 5'-diphosphate nucleotides by the following general mechanism: N1TP + N2DP 
N1DP + N2TP. Therefore, in the presence of these and nucleotidase enzymes, once in the extracellular environment, ATP or UTP metabolism may be both catabolic and anabolic. How these enzymes function together to produce different nucleotide metabolites is important, since P2 receptor subtypes are differentially selective for both di- and triphosphate adenine and uridine nucleotides.
The presence of ecto-nucleoside diphosphokinase (ecto-NDPK) enzymes on osteoblasts and the consequent interconversion of nucleotides have not been determined. In the study reported here, we have investigated ATP release by human osteoblasts and aimed to establish the fate of nucleotides once in the extracellular space to further determine the mechanisms regulating P2 receptor activation in bone cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Stocks of adherent SaOS-2 cells were cultured in 9 cm Petri dishes in DMEM, supplemented with 10% FCS, 50IU/mL penicillin, 50 µg/mL streptomycin, and 2 mM L-glutamine (referred to as complete DMEM). Human bone-derived cells (HBDCs) were isolated and cultured from human bone as described previously (34)
. Bone specimens in sterile saline were transferred to DMEM. The bone was cut into small pieces with a scalpel, then marrow cells were removed with several washes in medium. Bone pieces were cultured in 9 cm Petri dishes in complete DMEM. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 until confluent. Cells were detached using warm trypsin/EDTA; 1.5 x 105 cells were seeded into wells of 12-well plates and left in culture for 2 days (or until confluent) before ATP measurement experiments were performed.
Lactate dehydrogenase measurements
Cells were seeded at varying densities from 100 to 1,000,000 cells per well of a 12-well plate in complete DMEM, and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 for 24 h. Medium was replaced with 0.5 mL serum-free DMEM, after washing with warm PBS. Plates were incubated for another hour, after which 2 x 50 µL samples of cell culture medium were collected from each well and assayed for LDH activity using the CytoTox96 nonradioactive cytotoxicity assay kit from Promega. A standard curve was generated with known numbers of lysed cells. The resulting color change was read at 490 nm.
Measurement of extracellular ATP
Complete DMEM was removed from confluent SaOS-2 cells grown in 12-well plates and, after washing with warm PBS, replaced with 0.5 mL serum-free DMEM. Plates were incubated for another hour to ensure medium temperature was 37°C and to allow for degradation of any ATP that may have been released due to the medium change. Nucleotides or antagonists were introduced to the cells; P2Y1 and P2Y12 antagonists were introduced 2 min before nucleotide agonists and incubated with the cells at 37°C. Where more than one nucleotide agonist was introduced to cells, this was done simultaneously. 200 µL samples of cell culture medium were immediately collected into 5 mL round-bottomed polystyrene tubes and 20 µL of NMR containing luciferin and luciferase was added. The tube was placed in a Berthold Tube Luminometer (LB 955 Berthold, Wildbad, Germany) and readings were taken every second for 5 s; light emission was detected by a photon counter covering the spectral range from 380 to 630 nm. Average values were calculated and recorded as relative light units. Where nucleotides and/or antagonists were added to cell culture medium, ATP in identical samples was measured where nucleotides and/or antagonists had been added to serum-free culture medium alone. These values were subtracted from the cell culture medium sample values to account for ATP contamination of the nucleotides and antagonists (with the exception of Fig. 9A, B
). For Fig. 9A, B
, nucleotides were added to 1 mL of serum-free culture medium covering confluent SaOS-2 cells or 1 mL of serum-free culture medium alone, respectively. 40 µL samples of medium were taken every 10 min, immediately collected into 5 mL round-bottomed polystyrene tubes, and 4 µL of NMR was added. ATP in the samples was measured as outlined above. Finally, ATP standard curves were generated; from these, ATP concentrations in the cell culture medium were determined.
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RNA isolation and cDNA synthesis
Total RNA was isolated using TRI-REAGENTTM, which is based on a method first proposed by Chomczynski and Sacchi (35)
. Before first strand cDNA (cDNA) synthesis, RNA was DNase-treated with RNase-free DNase1 (35 U/mL) for 60 min at 37°C. DNase was inactivated by heating at 70°C for 15 min and the RNA precipitated for at least 1 h at –20°C in 3 volumes 100% ethanol and 0.1 volumes sodium acetate, pH 5.2. RNA was quantified by measuring absorbance at 260 nm on a Genequant microspectrophotometer; 5 µg of DNase-treated total RNA was used as a template for first-strand cDNA synthesis in a 20 µL reaction containing 0.5 µg oligo (dT), 0.5 mM dNTPs, 20 U of RNase inhibitor, 10 mM of dithiothreitol, 6 mM MgCl2, 40 mM KCl, 50 mM Tris-HCl, pH 8.3, and 200 U of Superscript II RT. The reaction mix was incubated at 42°C for 50 min and the reaction was stopped by heating at 70°C for 15 min. cDNA was stored at –20°C until required.
Polymerase chain reaction
25 µL reactions were performed on a MJ Research PTC-200 Peltier Thermal cycler. Reactions contained 1 µL of cDNA, 0.5 units of thermostable Taq DNA polymerase, 50 pmol each of sense and antisense primer, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, and 1.5 mM MgCl2 in 1x (final) reaction buffer. Standard reactions involved an initial 3 min denaturation step at 94°C, followed by the amplification step (94°C for 10 s, annealing Tm for 30 s, extension for 30 s at 72°C) for 40 cycles, plus a final 5 min extension step at 72°C. For analysis of DNA, PCR products were loaded onto 1% agarose gels containing 0.3 µg/mL ethidium bromide. Gels were run at 90–100 mA and the position of DNA in the gel was visualized by exposure to UV light. After excision of PCR product bands from agarose gels, Qiaex II gel extraction kits were used to extract and purify the product from the remaining agarose, according to the manufacturer’s protocol. Purified products were identified by sequence analysis. The primer sequences, Tms and product sizes are shown in Table 1.
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Immunocytochemistry
SaOS-2 and HBDCs cells were grown on 13 mm coverslips and fixed in 5% formalin. Coverslips were immersed in 1% (v/v) H2O2/H2O for 30 min, washed in PBS, then covered with 5% (v/v) polypep/PBS for an additional 30 min. This was removed and coverslips were incubated with 2 µg/mL monoclonal antibodies specific for nm23-H1 in 1% (w/v) BSA/PBS for 1 h at room temperature (RT) in a humidified Petri dish. Negative controls were incubated with 1% (w/v) BSA/PBS alone. After further PBS washes, coverslips were incubated with appropriate dilutions of HRP-coupled goat anti-mouse secondary antibody in 1% (w/v) BSA/PBS for 1 h at RT. The coverslips were washed in PBS and staining was developed using di-amino-benzidine (DAB). After rinsing in tap water, counterstaining with hematoxylin, and dehydration though a series of alcohols and xylene, coverslips were mounted onto microscope slides with DPX and examined by microscopy.
Statistical analysis
Statistical analysis was by ANOVA. The significance between groups was determined using a Tukey post test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). No LDH could be detected in similar medium samples (Fig. 1B
).
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ADP increases ATP concentration in cell culture medium
To determine whether osteoblasts were capable of converting nucleotides to ATP via ecto-nucleoside diphosphokinase activity, confluent SaOS-2 cells in 12-well plates were stimulated with various P2 receptor agonists; cell culture medium was collected and ATP concentration was calculated. Figure 2
shows that although addition of 10 µM ATP, 2MeSADP, UDP, UTP, or GTP had no effect on cell culture medium ATP concentration, introduction of 10 µM ADP to SaOS-2 cells resulted in an increase of 
35 nM ATP in the medium.
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SaOS-2 cells express P2Y1 and P2Y12 receptor mRNA
To determine whether receptor activation may have caused ATP release, PCR reactions were performed on cDNA generated from RNA isolated from two confluent 9 cm dishes of SaOS-2 cells used in the ATP measurement experiments. Primer pairs were designed specifically to P2Y1 and P2Y12 receptors, since ADP is the preferential agonist at P2Y1 and P2Y12 receptor subtypes and is not thought to be active at other P2 receptor subtypes (Table 1)
. Both P2Y1 and P2Y12 mRNAs were detected in these SaOS-2 cell cDNA samples (Fig. 3
).
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P2Y1 and P2Y12 antagonists have no effect on ADP-induced increase in cell culture medium ATP concentration
To discount the ADP-induced increase in cell culture medium ATP concentration as a specific effect of P2Y1 or P2Y12 activation, P2Y1 and P2Y12 antagonists (MRS2179 10 µM and AR-C69931MX 1 µM, respectively) were incubated at 37°C with confluent SaOS-2 cells in 12-well plates 2 min before addition of 10 µM ADP. Measurement of ATP in the cell culture medium revealed that neither antagonist, alone or in combination, had any effect on the ADP-induced increase in ATP (Fig. 4
).
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GTP dose-dependently increases ATP in SaOS-2 cell culture medium in the presence of ADP
If ecto-enzymes expressed by SaOS-2 cells were converting ADP to ATP, as has been reported in other cell types (30
31
32
33)
, available phosphates were likely to be a limiting factor for this conversion in the serum-free medium. Therefore, 20 µM GTP and 10 µM ADP were introduced simultaneously to confluent SaOS-2 cells. Addition of ADP alone again resulted in low nM ATP concentrations in the medium, but when ADP and GTP were added together, 4 µM ATP was measured in the cell culture medium (Fig. 5
A). GTP alone had no effect on ATP concentration. ATP increased dose-dependently when increasing concentrations of GTP were introduced to SaOS-2 cells with ADP (Fig. 5B
), and this dose-response reached a plateau at 100 µM GTP, where just under 10 µM ATP was measured in the cell culture medium (Fig. 5B
).
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UDP dose-dependently decreases ATP in SaOS-2 cell culture medium in the presence of ADP
The measurement of ATP after introduction of ADP and GTP to SaOS-2 cells strongly suggested that these cells did express an ecto-nucleoside diphosphokinase. To further prove the presence of these enzymes, we simultaneously introduced various combinations of nucleotides to confluent SaOS-2 cells or HBDCs. Since UDP is also known to be a substrate for ecto-NDPK, addition of high concentrations of UDP inhibits ADP from being used as a substrate when ADP is present at much lower concentrations (36
, 37)
. Figure 6
A shows that again addition of 10 µM ADP resulted in low nM ATP concentration in the medium, whereas GTP and UDP alone had no effect. Addition of 10 µM ADP and 20 µM GTP resulted in 4 µM ATP in the culture medium of both cell types, whereas the combinations of ADP and UDP and GTP and UDP had no effect. Most importantly, however, introduction of 10 mM UDP in combination with ADP and GTP completely prevented the formation of ATP in both SaOS-2 and HBDCs medium. This inhibition of ATP formation by UDP was dose dependent (Fig. 6B
), providing evidence to suggest competition of ADP and UDP for ecto-NDPK activity.
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SaOS-2 and HBDCs express NDPK/nm23-H1 mRNA
To verify that SaOS-2 and HBDCs express NDPKs, we performed PCR reactions on the same SaOS-2 cell cDNA samples used in Fig. 3
and on HBDC cDNA, using primer pairs designed specifically to NDPK/nm23-H1 (Table 1)
. NDPK/nm23-H1 mRNAs were detected in SaOS-2 cell cDNA samples and in both HBDC cDNA samples (Fig. 7
).
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NDPK/nm23-H1 protein is expressed on the extracellular SaOS-2 and HBDC cell membrane
Antibodies specific for NDPK/nm23-H1 were used in immunocytochemistry to identify the presence of this protein on the extracellular surface of SaOS-2 and HBDC cells. Positive staining was observed on both cell types; Fig. 8
A shows a typical field of view of positively stained HBDCs. Figure 8B
shows stained SaOS-2 cells. No staining was detected when the primary antibody was omitted (Fig. 8C
shows HBDCs, Fig. 8D
shows SaOS-2 cells).
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SaOS-2 cells express both ATP-forming and ATP-degrading ecto-enzymes
To determine how ecto-nucleotidases and ecto-NDPKs together affect ATP metabolism, ATP concentration in the cell culture medium of confluent SaOS-2 cells was measured for 2 h after addition of 10 µM ATP and 10 mM UDP, alone or in combination. ATP alone degraded more slowly than ATP plus UDP together (Fig. 9
A), demonstrating that inhibition of ecto-NDPK activity by UDP resulted in an overall lower concentration of total ATP to be degraded. Although ecto-nucleotidase activity was predominant over ecto-NDPK activity, there was a significant amount of ‘recycling’ of ATPADP. To confirm that this degradation was enzyme-mediated, identical experiments were performed in cell culture medium alone (Fig. 9B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence has been presented for the constitutive release of ATP from osteoblast cells. ATP was detected in SaOS-2 cell culture medium, and this was correlated with cell number (Fig. 1A
). When ATP was added to SaOS-2 cells, however, it was completely degraded within 120 min (Fig. 9A
), indicating that the maintenance of extracellular ATP concentrations by osteoblasts represents a net balance between constitutive release and subsequent hydrolysis and interconversion. ATP concentrations measured in the medium were probably below threshold stimulation for P2 receptors, but it is likely these measurements greatly underestimated the concentration that may be found in vivo due to the dilution factor of the relatively large volume of medium bathing the cells. In fact, a number of studies have confirmed that ATP concentration near cell surfaces is high enough to activate P2 receptors (38
, 39)
, and in addition to the release of ATP from osteoblast cells in bone, other cells in the local microenvironment are likely to contribute to the overall concentration of ATP in the extracellular compartment. The physiological release of ATP from nerves and vascular cells has been well documented (reviewed in ref 40
). The possibility that a proportion of total ATP concentration in the medium of SaOS-2 cells was due to membrane damage from a small fraction of the cells was discounted by the finding of no LDH in the culture medium (Fig. 1B
). Current evidence supports that constitutive ATP release is a cell-regulated process that may involve ion channels (41
, 42)
and/or phosphatidylinositol 3-kinase (43)
. The involvement of members of the ATP binding cassette (ABC) transporter superfamily in the regulation of ATP release has been speculated for some time, but this theory has recently begun to be doubted (44
, 45)
. Constitutive release of ATP has been suggested as a mechanism for regulating nucleotide concentration at the inner surface of the plasma membrane (46)
; alternatively, this release may be part of an autocrine/paracrine signaling system that results in P2 receptor activation.
Addition of ADP to SaOS-2 cells resulted in detection of low concentrations of ATP in the cell culture medium (Fig. 2)
, providing the first indication of enzyme-mediated nucleotide synthesis by these cells. This experiment was reproduced in SaOS-2 cell conditioned medium alone, with no detection of ATP formation (data not shown), indicating that enzyme activity was membrane bound and not soluble. Production of ATP in the absence of added nucleotides suggested the presence of triphosphates in the medium. These triphosphates could not have been ATP since comparable measurements would have been made in vehicle samples, but were most likely UTP, which has also been shown to undergo constitutive cellular release (47)
. The possibility of ADP-induced receptor activation in mediating ATP release was discounted by use of specific antagonists. ADP is the preferential agonist at P2Y1 and P2Y12 receptor subtypes, and mRNAs encoding both subtypes were found to be expressed by SaOS-2 cells (Fig. 3)
. Incubation of cells with P2Y1 and P2Y12 specific antagonists and ADP, however, did not prevent ATP formation. Addition of 10 µM ADP and 20 µM GTP hugely increased ATP concentration in the culture medium (Fig. 5A
), revealing evidence for a transphosphorylating activity of SaOS-2 cells. Approximately only half of the ADP was converted to ATP, suggesting 20 µM GTP was still a limiting factor in this conversion; 100 µM GTP was required to convert nearly all of the ADP to ATP (Fig. 5B
).
Although formation of ATP from ADP can be effected by adenylate kinase (2ADPATP+AMP), no evidence for the occurrence of this reaction was detected. Total inhibition of conversion of ADPATP by UDP (Fig. 6A
) identified an ecto-NDPK as the enzyme responsible for this transphosphorylation, since it is well known that ecto-NDPK can be characterized by its ability to phosphorylate purine and pyrimidine dinucleotides, as well as its inhibition by high concentrations (mM) of pyrimidine dinucleotide substrate (36
, 37)
. UDP also inhibited the transphosphorylation of ADPATP by HBDCs, with results almost identical to those produced from SaOS-2 cells (Fig. 6A
). Although it was not possible to perform all experiments using HBDCs due to their limited supply and slow rate of growth, the data unequivocally demonstrated the presence of primary osteoblast ecto-NDPK activity and allows us to presume that findings from SaOS-2 cells accurately reflect the activity of HBDCs. Ecto-NDPK activity measured in SaOS-2 cells was found to have greater affinity for ADP than UDP: when identical concentrations of these nucleotides were introduced simultaneously with GTP, ATP formation decreased by only 20% compared with ADP and GTP alone (Fig. 6B
). These findings differ from a study of ecto-NDPK activity of 1321N1 astrocytoma cells that reported similar affinity for ADP or UDP (48)
.
The human gene nm23-H1 has been cloned and shown to encode for a 17 kDa protein with NDPK activity (49)
. It has not yet been determined whether this enzyme accounts for all or only a fraction of total ecto-NDPK activity in any cell type, but both nm23-H1 mRNA and protein were detected in SaOS-2 cells and HBDCs (Figs. 7
and 8)
, demonstrating that the observed ecto-NDPK activity was due either entirely or partly to its actions and providing molecular evidence to support the functional studies.
It has been reported that osteoblasts express ecto-nucleotidase enzymes (27)
; we therefore investigated the combined actions of these and ecto-NDPK enzymes. ATP degradation occurred at a greater rate when NDPK enzymes were inhibited (Fig. 9A
), suggesting that recycling of ATPADP occurred when both types of enzyme were active; therefore, ecto-NDPK activity provides an extracellular ATP regenerating system. One function of this may be to produce an increased supply of the high-energy phosphate bond of ATP that may be used for cellular processes such as phosphorylation of extracellular proteins. The data shown in Fig. 9A
also reveal how the combined actions of ecto-NDPK and ecto-nucleotidase enzymes may act to maintain specific concentrations of extracellular nucleotides to ensure activation of certain P2 receptors. The transphosphorylating actions of ecto-NDPK enzymes allow numerous receptor subtypes, preferentially activated by either adenine or uridine nucleotides, to be stimulated. Specific enzymatic expression and physiological circumstances may locally influence the direction of nucleotide synthesis, hydrolysis and transphosphorylation.
The discovery of ecto-NDPK expression by osteoblasts induces reevaluation of previous studies involving addition of nonstable dinucleotides to osteoblast cultures. Effects apparently caused by addition of ADP may not necessarily be due to actions of this nucleotide, as we have demonstrated here its rapid conversion to ATP. Although this will subsequently undergo hydrolysis back to ADP, these changes are highly transient and the potential presence of ADP, ATP, UDP, and UTP after ADP addition, all of which are active at different P2 receptor subtypes, means it would be inaccurate to attribute only ADP activation of P2Y1 or P2Y12 receptors to the induced effect. Similar caution should be given to other nucleotides, stressing the importance of using nonhydrolyzable nucleotide compounds in P2 receptor studies.
P2 receptor activation in bone provides important signals to osteoblast and osteoclast cells to affect their behavior. Control of the duration of nucleotide signal may allow regulation of intercellular signaling in bone, since calcium waves across neighboring osteoblasts can be initiated by mechanically induced nucleotide release (50)
, and this communication has also been observed in osteoclasts (51)
. Osteoblasts are more responsive to parathyroid hormone (PTH) when P2 and PTH receptors are costimulated (52
, 53)
. This phenomenon may provide some explanation for the localized nature of bone remodeling despite the systemic actions of PTH, one of the most influential factors on this process. Localized release of ATP and control of its hydrolysis, synthesis, and transphosphorylation by osteoblasts may provide a highly regulated system for modulating focal remodeling events. Similarly, osteoclast formation and resorption, both of which are increased by P2 receptor activation (10
, 15
16
17
18)
, may be affected by release and subsequent interconversion of nucleotides by osteoblasts. This nucleotide release and metabolism may also represent an autocrine signaling system whereby osteoblasts are able to modulate their own rate of proliferation (9)
, DNA synthesis (6)
, or RANKL expression (10)
.
Here we have identified an ecto-NDPK activity associated with SaOS-2 and primary osteoblast cells that, in the presence of a 
-phosphate donor, promotes formation of ATP or UTP from their corresponding diphosphate nucleotides. The coexistence of two opposite metabolic pathways of extracellular nucleotide hydrolysis and synthesis may represent a mechanism for regulating cell-specific responses to circulating adenine and uridine nucleotides and the termination of their purinergic function. Identification of phospho-transfer reactions between adenine and uridine nucleotides by osteoblast cells provides further understanding for interpretation of the physiological roles of these nucleotides as signaling molecules in bone.
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ACKNOWLEDGMENTS |
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Received for publication October 22, 2002. Accepted for publication April 16, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B. Bone 31,582-590[Medline]
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