(Received for publication, May 5, 1997, and in revised form, May 30, 1997)
From the Departments of Pharmacology and
§ Medicine, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599 and the ¶ Research
Group of Hungarian Academy of Sciences, Budapest, Hungary
The P2Y4 receptor is
selectively activated by UTP. Although addition of neither ATP nor UDP
alone increased intracellular Ca2+ in 1321N1 human
astrocytoma cells stably expressing the P2Y4 receptor,
combined addition of these nucleotides resulted in a slowly occurring
elevation of Ca2+. The possibility that the stimulatory
effect of the combined nucleotides reflected formation of UTP by an
extracellular transphosphorylating activity was investigated.
Incubation of cells with [3H]UDP or [3H]ADP
under conditions in which cellular release of ATP occurred or in the
presence of added ATP resulted in rapid formation of the corresponding
triphosphates. Transfer of the -phosphate from [
-33P]ATP to nucleoside diphosphates confirmed that
the extracellular enzymatic activity was contributed by a nucleoside
diphosphokinase. The majority of this activity was associated with the
cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme.
Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the
primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP
also were determined for 1321N1 cells. All four nucleotides were
hydrolyzed with similar Km and
Vmax values. Kinetic analyses of the
ecto-nucleoside diphosphokinase and ecto-nucleotidase activities
indicated that the rate of extracellular transphosphorylation exceeds
that of nucleotide hydrolysis by up to 20-fold. Demonstration of the
existence of a very active ecto-nucleoside diphosphokinase together
with previous observations that stress-induced release of ATP occurs
from most cell types indicates that transphosphorylation is
physiologically important in the extracellular metabolism of adenine
and uridine nucleotides. Since the P2Y receptor class of signaling
proteins differs remarkably in their respective specificity for adenine
and uridine nucleotides and di- and triphosphates, these results
suggest that extracellular interconversion of adenine and uridine
nucleotides plays a key role in defining activities in
nucleotide-mediated signaling.
The importance of adenine nucleotides as extracellular signaling molecules is well established (1, 2). ATP and/or ADP are released in a regulated fashion from neurons, platelets, and other cells and interact with two major classes of cell surface receptors, the ligand-gated P2X receptors and the G protein-coupled P2Y receptors (3-5). These receptors, which are encoded by at least a dozen different genes, in turn promote an exceptionally broad range of functional responses. Although physiologically important release of uridine nucleotides is less well defined, the identification of at least three P2Y receptors that are selectively activated by low concentrations of UTP or UDP is consistent with an important extracellular signaling role for pyrimidines (5, 6).
Hydrolysis by ecto-nucleotidases provides a mechanism whereby the physiological effects of extracellular nucleotides are terminated (2, 7-9). Degradation of ATP and ADP also apparently serves as a major source of extracellular adenosine, which in turn activates A1, A2, and A3 adenosine receptors (10). The enzymatic species involved in hydrolysis of extracellular nucleotides have not been unambiguously defined, although certain ATP-diphosphohydrolases exhibiting kinetic properties consistent with those of physiologically relevant ecto-nucleotidases have been purified and/or cloned (11, 12). The possibility that other types of ectoenzymes contribute to the metabolism and/or interconversion of extracellular adenine and uridine nucleotides has not been considered extensively.
Nucleoside diphosphokinase
(NDPK)1 catalyzes the
transphosphorylation of nucleoside diphosphates utilizing
nucleoside triphosphates as the -phosphate donor (13). Intracellular
NDPK fulfills a crucial role in maintaining the high energy phosphate
bond in ATP as part of the citric acid chain. NDPK also has been
proposed to play a major role in the cytosolic synthesis of nucleoside triphosphates in addition to ATP and in maintaining a relative balance
in the concentrations of nucleoside triphosphates. Human nm23
genes encode for nucleoside diphosphokinases (14, 15), and an
inverse relationship exists between nm23 expression and metastatic potential (16, 17).
In contrast to its well established significance in intermediary
metabolism, the potential location and function of NDPK as an
extracellular enzyme involved in the transfer of terminal phosphates between extracellular nucleotides has not been determined. Therefore, we have tested this possibility using 1321N1 human astrocytoma cells
stably expressing the P2Y4 receptor which we show is
selectively activated by UTP. Extracellular conversion of UDP to UTP
has been measured in the presence of ATP, and P2Y4
receptor-promoted elevation of intracellular Ca2+ has been
quantitated as a functional measure of this conversion. Accordingly, we
have identified an ecto-NDPK activity associated with 1321N1 cells
that, in the presence of a -phosphate donor, promotes formation of
UTP or ATP from their corresponding diphosphate nucleotides. The
activity of this enzyme exceeds that of the ecto-nucleotidase activity
by 20-fold. Thus, NDPK activity promotes active interchange of
-phosphates between endogenous adenine and uridine nucleotides on
the surface of 1321N1 cells, and this interconversion has significant implications in establishing selectivity of activation of P2Y receptors
which differ markedly in their nucleoside diphosphate and triphosphate
specificity.
Wild type 1321N1 human astrocytoma cells and 1321N1 cells infected with retrovirus harboring the P2Y4 receptor sequence (provided by Dr. R. Nicholas and Dr. J. Schachter) were cultured in DMEM-high glucose (DMEM-H) medium supplemented with 5% fetal bovine serum and antibiotics as described (18). The cells were grown to confluence on 24-well plastic plates (except where indicated otherwise) for nucleotide metabolism studies or on 25-mm glass coverslips previously coated with 0.3 mg/ml vitrogen for calcium measurements.
Measurement of Intracellular Ca2+P2Y4 receptor-expressing cells were incubated with 3 µM Fura-2/AM for 30 min at 37 °C. After the loading period, the cells were bathed in 0.4 ml of Ringer solution (130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.4) and mounted in a microscope chamber. The fluorescence (>450 nm) of 30-50 cells was alternately determined at 340 and 380 nm excitation by a RatioMaster RM-D microscope fluorimetry system (Photon Technology Inc., Monmouth Junction, NJ) at room temperature. A Zeiss Axiovert 35 inverted microscope and Nikon UV-F 100×/1.30 glycerol immersion objective were used. After each experiment, the cells were lysed with 40 µM digitonin, and the background fluorescence was determined by quenching with 4 mM manganese. The background-corrected ratio values (340/380) were calibrated by using the formula originally proposed by Grynkiewicz et al. (19). Rmax, Rmin, and Kd values were determined by using 1 µM Fura-2 free acid and a series of Ca2+ buffers.
Metabolism of Nucleotides by 1321N1 CellsThe cells were
washed three times with serum-free DMEM medium and bathed in 0.5 ml of
DMEM-H/HEPES, pH 7.4. Incubations were initiated by the addition of
drugs and terminated by transferring the medium to a tube containing 50 µl of 50 mM EDTA and subsequently boiled for 1 min. The
samples were maintained at 20 °C prior to HPLC analysis.
1321N1 cells were incubated in the presence of ATP and with various concentrations of ADP or UDP and 0.1-0.5 µCi of [3H]ADP or [3H]UDP to provide a range of specific radioactivities of the nucleotides. The conversion of [3H]ADP to [3H]ATP and [3H]UDP to [3H]UTP was determined by HPLC analysis.
Synthesis of [3H]ADP and [3H]UDP[3H]ADP and [3H]UDP were obtained from their respective 3H-labeled nucleotide triphosphates by a hexokinase-catalyzed reaction as described previously (20). Briefly, 20-50 µCi of either [3H]ATP or [3H]UTP (40-50 Ci/mmol) were incubated with 10 units/ml hexokinase for 30 min at 37 °C in 0.2 ml of DMEM-H/HEPES, pH 7.4. After incubations, the samples were boiled for 1 min to eliminate the hexokinase activity. Full conversion of [3H]NTP to [3H]NDP was confirmed by HPLC.
Transfer of the1321N1 cells were washed and incubated as
above in 0.5 ml of DMEM-H/HEPES medium in the presence of 300 µM [-33P]ATP and 100 µM of
the various nucleoside diphosphates. The transfer of
[
-33P]ATP was determined by HPLC analysis.
Two sets of confluent 1321N1 cells grown on 24-well plates were washed three times and bathed in 0.5 ml of DMEM-H/HEPES. After 1 min at 37 °C the medium from one set of cells was collected, rapidly centrifuged to remove any detached cells, and transferred to a tube containing 0.5 µCi of [3H]ADP (50 nmol) and ATP (150 nmol). [3H]ADP and ATP also were added to the second set of cells, and the NDPK activity of the medium and cells was assayed for 1 min. Total cellular activity of NDPK was also determined. Cells from 4 wells cultured in a separate 24-well plate were trypsinized, washed twice, resuspended in 2 ml of 50 mM ice-cold Tris, pH 7.2, and sonicated. NDPK activity was assayed in a 1:30 dilution of the cell sonicate.
Measurement of Ecto-nucleotidase Activity1321N1 cells were washed and incubated in 0.5 ml of DMEM-H/HEPES in the presence of 0.5 µCi and the indicated concentrations of [3H]ADP, [3H]UDP, [3H]ATP, or [3H]UTP. Since preliminary experiments indicated that 1321N1 cells avidly transport adenosine but not uridine, studies of [3H]ADP and [3H]ATP hydrolysis were carried out in the presence of the nucleoside transport inhibitor dipyridamole (100 µM). Dipyridamole did not affect the rate of hydrolysis of ATP or ADP by 1321N1 cells (data not shown).
HPLC Separation of NucleotidesNucleotides were separated
and quantified by HPLC (Shimadzu) via a Hypersil-SAX column (Bodman,
Aston, PA) using a 30-min linear gradient developed from 5 mM NH4H2PO4, pH 2.8, to
0.75 M NH4H2PO4, pH
3.7. Absorbance at 264 nm was monitored with an SPD-10A UV detector
(Shimadzu), and radioactivity was determined on-line with a Flo-One
Radiomatic detector (Packard, Canberra, Australia) as described
previously (21).
All nucleoside triphosphates were purchased from
Pharmacia (Uppsala, Sweden). Hexokinase, ouabain, tetramisol,
Ap5A, ADP, UDP, GDP, and CDP were from Boehringer Mannheim.
Dipyridamole and TDP were from Sigma. Vitrogen was from Collagen Corp.,
Palo Alto, CA. Fura-2/AM and Ca2+ buffer were from
Molecular Probes (Eugene, OR). [3H]ATP (40 Ci/mmol),
[3H]UTP (50 Ci/mmol), and [-33P]ATP
(3000 Ci/mmol) were from Amersham.
Our initial evidence that extracellular NDPK activity is
associated with 1321N1 human astrocytoma cells emanated from studies of
the cloned P2Y4 receptor stably expressed in these cells.
We (18) and others (22, 23) originally reported that, although much
less potent than UTP (EC50 = 0.4 µM), ATP
(EC50 = 30 µM) nonetheless was an agonist in
15-20-min assays of inositol phosphate accumulation in
P2Y4 receptor-expressing 1321N1 cells. However, contrasting
results were obtained in studies of Ca2+ mobilization by
Nguyen et al. (24), and we have confirmed their results
here. Addition of 1 µM UTP to Fura-2-loaded
P2Y4 receptor-expressing 1321N1 cells resulted in rapid
mobilization of intracellular Ca2+ (Fig.
1, upper tracing). In
contrast, neither 1 µM UDP (in the presence of
hexokinase) nor 10 µM ATP elevated intracellular
Ca2+ levels. However, a small response to UDP was observed
if hexokinase was not added to the cells (Fig. 1, lower
tracing). Moreover, combined addition of 1 µM UDP
and 10 µM ATP resulted in a slowly occurring but
sustained increase in intracellular Ca2+ (Fig. 1,
lower tracing). Taken together these results suggested that
the stimulatory effects at the P2Y4 receptor of the
combined presence of UDP and ATP occurred as a consequence of formation of UTP by an endogenous transphosphorylating activity. Because 1321N1
cells readily release ATP upon mechanical stimulation (21), the small
effect of UDP in the absence of hexokinase might reflect formation of
UTP from endogenous ATP.
To determine whether NDPK activity could be detected more directly,
P2Y4 receptor-expressing cells were incubated with either 1 µM [3H]ADP or 1 µM
[3H]UDP in the absence or presence of 10 µM
ATP, and the formation of [3H]ATP or
[3H]UTP was assessed by HPLC. In the absence of added
ATP, the radiolabeled nucleoside diphosphates were partially converted
to the corresponding labeled nucleoside monophosphates, and no
formation of 3H-labeled nucleoside triphosphate was
detected (Fig. 2). In contrast, in the
presence of 10 µM ATP, [3H]ADP and
[3H]UDP were converted to their respective nucleoside
triphosphates with little evidence of formation of the monophosphate
species.
Conversion of nucleoside diphosphates to triphosphates by transfer of
-phosphate was confirmed in experiments carried out with
[
-33P]ATP. Incubation of 1321N1 cells with
[
-33P]ATP and UDP or GDP resulted in the rapid
formation of [
-33P]UTP or [
-33P]GTP,
respectively (Fig. 3). Thus, a highly
active NDPK activity is associated with the extracellular environment
of 1321N1 cells. This activity is not a result of overexpression of the
P2Y4 receptor in 1321N1 cells, since in the presence of 100 µM [3H]ADP and 300 µM ATP
similar formation of [3H]ATP was observed with wild type
cells (5.1 ± 0.7 nmol/106 cells/min),
P2Y2 receptor-expressing cells (4.4 ± 0.2 nmol/106 cells/min), and P2Y4
receptor-expressing cells (2.7 ± 0.6 nmol/106
cells/min).
Time course experiments indicated that in the presence of unlabeled
ATP, 10 µM [3H]ADP was rapidly
phosphorylated to [3H]ATP, and a maximal conversion
(approximately 60%) occurred within 2 min after drug addition to
1321N1 cells (Fig. 4A). Under
the same conditions the rate of formation of [3H]UTP was
slower than was the formation of [3H]ATP (Fig.
4A), and a maximal conversion of approximately 30% occurred
within 20 min. We have reported previously that relatively large
amounts of endogenous ATP are released from 1321N1 cells upon
mechanical stimulation (21). Addition of [3H]ADP or
[3H]UDP to mechanically stimulated 1321N1 cells also
resulted in rapid formation of [3H]ATP or
[3H]UTP (Fig. 4B), and the extent of formation
of ATP was greater than that of UTP. Observation of rapid conversion of
extracellular ADP to ATP and of UDP to UTP by intact 1321N1 cells under
conditions in which nucleotide released from cells serves as a
-phosphate donor suggests that NDPK activity is important at
pharmacologically relevant concentrations of nucleotides. For example,
in the presence of endogenously released ATP, addition of 10 µM UDP (to 0.5 ml of medium bathing 0.3 × 106 cells) resulted in accumulation of approximately 0.3 µM UTP (Fig. 4B), a concentration that
markedly activates the P2Y2 (21) and P2Y4 (18)
receptors.
The concentration dependence for ADP and UDP for promotion of NDPK
activity was determined in the presence of a near-saturating concentration (300 µM) of ATP. Assays were carried out
under apparently initial rate conditions where <10% of the
diphosphate was converted to the corresponding triphosphate. Conversion
of [3H]UDP to [3H]UTP by 1321N1 cells
occurred with Vmax(app) and
Km(app) values that were approximately
one-half and two-fold, respectively, the values obtained in analogous
experiments examining the conversion of [3H]ADP to
[3H]ATP (Fig. 5A
and Table I). UDP inhibited the
ATP-promoted formation of [3H]ATP from
[3H]ADP and ADP inhibited the ATP-promoted formation of
[3H]UTP from [3H]UDP (Table
II). Furthermore, 50 µM UDP
caused an approximately 3-fold shift to the right of the concentration
effect curve for ADP but had no effect on the
Vmax(app) for ADP (data not shown). The
concentration dependence for ATP also was determined in the presence of
a fixed concentration (100 µM) of [3H]ADP
(Fig. 5B). The observed
Km(app) for ATP was 4-7-fold higher
than the values determined for the nucleoside diphosphates (Table
I).
|
|
EDTA (5 mM) completely inhibited the conversion of [3H]ADP to [3H]ATP whereas 5 mM EGTA had no effect (data not shown). These results suggest that the ecto-NDPK activity is Mg2+-dependent. Ap5A, a well characterized inhibitor of adenylate kinase (25), had no effect on the conversion of [3H]ADP to [3H]ATP, suggesting that adenylate kinase activity does not contribute in the conversion of diphosphates to triphosphates by 1321N1 cells.
The substrate selectivity of the NDPK activity was determined for both nucleoside triphosphates and nucleoside diphosphates (Table III). GTP and ATP were equally effective in promoting conversion of [3H]ADP to [3H]ATP, whereas a significantly slower rate of phosphorylation of [3H]ADP was obtained with CTP (30-40% relative to ATP). GDP and ADP also were preferred acceptor substrates relative to CDP (Table III).
|
The localization of the extracellular NDPK activity also was
determined. Approximately 70% of the total extracellularly measured NDPK activity was associated with the cell surface of 1321N1 cells (Fig. 6). An approximate doubling of
medium NDPK activity occurred over a 60-min period after an extensive
wash (rapid change of the medium three times) of 1321N1 cells (data not
shown). Under no conditions did medium NDPK activity approximate that
of cell surface NDPK activity. The total cellular NDPK activity
(determined as described under "Materials and Methods") was
approximately 10-fold higher than the total activity associated with
the cell surface (data not shown).
The role of ecto-nucleotidases in the metabolism of extracellular
adenine nucleotides has been widely studied in various tissues. In
light of the ecto-NDPK activity found associated with 1321N1 cells,
comparative experiments were carried out measuring the rates of
hydrolysis of extracellular nucleoside tri- and diphosphates. Since
essentially all analyses of ecto-nucleotidases to date have focused on
the hydrolysis of adenine nucleotides, we also determined the relative
effectiveness of 1321N1 cells for hydrolysis of extracellular uridine
nucleotides. Extracellular [3H]ATP (1 µM)
was hydrolyzed (Fig. 7) by 1321N1 cells
with a half-time of approximately 25 min (in a volume of 500 µl on a
1-cm2 well of a 12-well dish). The time courses of
disappearance of UTP, ADP, and UDP were essentially superimposable with
that observed for ATP (Fig. 7). The similarity of adenine and uridine
nucleotide tri- and diphosphates as substrates for ecto-nucleotidase
activity was confirmed in kinetic experiments. Thus, the observed
Km and Vmax values for all
four molecules were very similar (Table IV). This nucleotidase activity was
unchanged in the presence of 2 mM ouabain (a P-type ATPase
inhibitor), 5 mM tetramisol (an alkaline phosphate
inhibitor), 10 mM NaN3 (a mitochondrial ATPase inhibitor), or 10 mM NaF (a nonspecific inhibitor of
phosphatase), but it was abolished by chelators of
Ca2+/Mg2+ (data not shown). The similarity of
the kinetic constants calculated for nucleotidase activity against the
four adenine and uridine nucleotides and the lack of inhibitory effects
of several ATPase and phosphatase inhibitors suggest that the
nucleotidase activity is an apyrase type, i.e. it is an
ATP-diphosphohydrolase (8, 9, 26). The results obtained in kinetic
analysis of the nucleotidase activity of 1321N1 cells also were
consistent with the data illustrated in Fig. 2, B and
C. That is, the NDPK activity associated with 1321N1 cells
would be expected to exceed the ecto-nucleotidase activity by up to
20-fold in the presence of low micromolar concentrations of nucleoside
diphosphate and an excess concentration of a -phosphate-donating nucleoside triphosphate.
|
This study identifies an ecto-NDPK activity associated with the
extracellular surface of 1321N1 cells. Transphosphorylating activity
apparently emanates from a single enzymatic species that utilizes
either ADP or UDP similarly well as substrates for formation of the
corresponding triphosphate. Although formation of ATP from ADP can be
effected by adenylate kinase (2ADP ATP + AMP), no evidence for the
occurrence of this reaction was detected. Moreover, the rate of
transfer of [
-33P]phosphate from extracellular ATP to
nucleoside diphosphates approximated the rates of conversion of
3H-labeled nucleoside diphosphates to nucleoside
triphosphates. Therefore, the majority of this conversion must occur
due to an extracellular NDPK activity.
The observed NDPK activity is not trivial. Indeed, kinetic analyses of the NDPK and nucleotidase activities indicated that at relatively low diphosphate concentrations the NDPK activity exceeds that of the extracellular nucleotidase activity by up to 20-fold. This calculated surfeit of NDPK activity relative to nucleotidase activity was confirmed directly. That is, addition of [3H]ADP or [3H]UDP to the medium of 1321N1 cells in the presence of ATP resulted in relatively large formation of the corresponding radiolabeled triphosphates with little evidence of conversion of the diphosphates to monophosphates.
The extracellular NDPK activity is largely, but not exclusively, found as a surface membrane-associated ectoenzyme rather than as an enzymatic activity in the extracellular medium. The identity of this extracellular NDPK is not yet clear. Two putative tumor suppressor genes, nm23-H1 and mm23-H2, have been cloned and shown to encode for approximately 17-kDa proteins (also called NDPK A and NDPK B) that exhibit NDPK activity (14, 15). It is unclear whether these enzymes account for all of the intracellular NDPK activity, and we have not yet addressed whether the ecto-NDPK activity is the same species as the previously molecularly identified forms of NDPK activity.
Extracellular NDPK has functional significance in 1321N1 cells.
Although kinetic analyses required addition of known amounts of
nucleoside triphosphate to the medium, substantial conversion of
radiolabeled diphosphate to triphosphate occurred under conditions, e.g. a change of medium, in which ATP is released from
1321N1 cells (21). Thus, the released triphosphate readily serves as a
-phosphate donor for exogenously added nucleoside diphosphates. Such
interconversion of nucleotides also has a major influence on
pharmacological effects observed with exogenously applied nucleotide agonists. Thus, as was illustrated in Fig. 1, UDP in the absence but
not in the presence of hexokinase raised Ca2+ in
P2Y4 receptor-expressing 1321N1 cells. Coaddition of UDP
with ATP resulted in a marked Ca2+ response. Thus,
observations (22, 24) indicating that UDP was an agonist at the
P2Y4 receptor expressed in 1321N1 cells likely were due to
conversion of UDP to UTP in the presence of released endogenous ATP
during the 15-20-min measurements of inositol phosphate accumulation.
Similarly, we have shown that the full agonist effects that we and
others originally reported for UDP and ADP at the P2Y2
receptor could be at least in part due to NDPK-promoted conversion of
these diphosphates to their corresponding triphosphates (18, 21), which
are potent full agonists at the P2Y2 receptor. We also have
reported that since hexokinase in the presence of glucose converts ATP
and UTP to their corresponding diphosphates, this enzyme can be
included in the medium in experiments designed to determine the
pharmacological effects of nucleoside diphosphates (18, 20), a strategy
that blocked the UDP effect on Ca2+ in P2Y4
expressing cells (Fig. 1). Since 1321N1 cells represent the principle
null cell line in which P2Y receptors have been expressed, the presence
of a heretofore unrecognized ecto-NDPK activity on these cells may
explain in large part the discrepant results reported by various
laboratories in studies of the pharmacological selectivities of these
receptors.
Not only is the ecto-NDPK similarly active against adenine and uridine nucleotide substrates, but our results indicate that the ecto-nucleotidase activity of 1321N1 cells also hydrolyzes uridine nucleotides at rates similar to that observed with adenine nucleotides. Thus, the extracellular hydrolytic machinery that has been widely studied and established as an important component of the extracellular adenine nucleotide signaling apparatus likely has a similarly important role in terminating the action of extracellular uridine nucleotides. Identification of an ecto-NDPK activity that is equally active against adenine and uridine nucleotides now adds a second level of complexity in understanding the physiological roles of adenine and uridine nucleotides as extracellular signaling molecules.
Although the data presented here have arisen entirely from studies of 1321N1 cells, the extracellular conversion of diphosphates to triphosphates is not restricted to this tumor cell line. For example, we have observed similar extracellular interconversion of nucleotides in studies of polarized human airway epithelial cells that maintain many of the phenotypical characteristics associated with these cells in vivo (20). Whether extracellular NDPK activity is expressed in a cell- or tissue-specific manner will need to be established. The occurrence of extracellular levels of enzyme activity similar to those observed with 1321N1 cells would have major physiological significance in regulating the extracellular signaling properties of adenine and uridine nucleotides.