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MediCity Research Laboratory, University of Turku and National Public Health Institute, FIN-20520, Turku, Finland
1Correspondence: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, FIN-20520, Turku, Finland. E-mail: gennady.yegutkin{at}utu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ADP ATP. The following two groups of
nucleotide-converting ecto-enzymes were identified based on inhibition
and substrate specificity studies: 1)
ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase;
2) ecto-nucleotide kinases, adenylate kinase, and
nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase
possessed the highest activity, as revealed by comparative kinetic
analysis, and was capable of using both adenine and nonadenine
nucleotides as phosphate donors and acceptors. The transphosphorylation
mechanism was confirmed by direct transfer of the 
-phosphate from
[-32P]ATP to AMP or nucleoside diphosphates and by
measurement of extracellular ATP synthesis using luciferin-luciferase
luminometry. The data demonstrate the coexistence of opposite,
ATP-consuming and ATP-generating, pathways on the cell surface and
provide a novel mechanism for regulating the duration and magnitude of
purinergic signaling in the vasculature.—Yegutkin, G. G.,
Henttinen, T., Jalkanen, S. Extracellular ATP formation on vascular
endothelial cells is mediated by ecto-nucleotide kinase activities via
phosphotransfer reactions.
Key Words: adenine nucleotides • ecto-enzymes • hydrolysis • interconversion • vasculature
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. Once released,
these nucleotides interact with two major classes of purinergic
receptors, G-protein-coupled (P2Y) and ligand-gated (P2X), to modulate
diverse cellular functions in an autocrine or paracrine fashion
(6
, 7)
. Finally, adenine nucleotides are degraded to
adenosine by members of several ecto-enzyme families, such as E-type
ecto-ATPases (EC 3.6.1.15)/ATP-diphosphohydrolases (ATPDases; EC
3.6.1.5) (1
, 8
, 9)
, nucleotide pyrophosphatases (EC
3.6.1.9)/phosphodiesterases I (EC 3.1.4.1) (5
, 9
10
11)
, and
5'-nucleotidases (EC 3.1.3.5) (1
, 12
, 13)
. The generated
adenosine can serve as an agonist for its own P1 receptors or be
imported into the cell by specific Na+-dependent
nucleoside transporters to replenish intracellular purine nucleoside
and nucleotide pools (6
, 7)
.
Some of the classical intracellular nucleotide-converting enzymes can
also be expressed on the cell surface, such as nucleoside diphosphate
(NDP) kinase (EC 2.7.4.6), normally maintaining the balance of ribo-
and deoxyribonucleoside triphosphates in the cell (14)
,
and ATP synthase (EC 3.6.1.34), otherwise known as
F0F1-ATPase or
H+-ATPase, which usually catalyzes the
mitochondrial ATP synthesis from ADP and orthophosphate using
proton-motive force (15)
. The possibility of extracellular
interconversion of nucleoside triphosphates (NTP) and NDP through NDP
kinase reaction has been suggested earlier for several cell types
(16
17
18)
, including cultured aortic endothelial cells
(19)
. ATP synthase also occurs as an ecto-enzyme on human
umbilical vein endothelial cells (HUVEC) (20)
and several
tumor cell lines (21)
, and functions to bind angiostatin
or facilitate lymphocyte-mediated cytotoxicity.
In the vasculature, the importance of extracellular adenine nucleotides
is defined on the one hand by direct effects on regional blood flow,
platelet aggregation, and the function of lymphocytes and granulocytes,
and on the other hand by further nucleotide degradation to adenosine,
which can also regulate vascular processes with different, often
opposite effects (2
, 7
, 22)
. Vascular endothelial
cell-associated E-type ATPases together with 5'-nucleotidase provide
the major effector system whereby purinergic effects of ATP and ADP are
terminated in the bloodstream (22
23
24
25)
; the biochemistry
and cell biology of these ecto-enzymes have been extensively
characterized. The enzymes of E-type ATPase family are glycoproteins
with two predicted transmembrane domains at the amino and carboxyl
terminus and a large extracellular loop containing the five ‘apyrase
conserved regions’ relevant for catalytic activity (9
, 26
, 27)
. Another ecto-enzyme 5'-nucleotidase, otherwise known as
CD73, is attached to the membrane matrix via a
glycophosphatidylinositol (GPI) anchor (12
, 13)
and, like
other GPI-anchored proteins, is presumably located in specific
detergent-insoluble membrane microdomains (rafts) enriched in
glycosphingolipids and cholesterol and possessing relatively high
lateral mobility (28
, 29)
. Compared to the
nucleotidase-mediated degradation pathway, adenine nucleotide synthesis
on the endothelial surface has so far received little attention.
In the present work, we took advantage of a thin-layer chromatography (TLC) assay to establish the main exchange activities of exogenous 3H-labeled adenine nucleotides on the surface of cultured HUVEC. This approach allowed us to identify and characterize two different types of ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and NDP kinase, participating in concert with ecto-nucleotidases in the interconversion of extracellular nucleotides. The coexistence of two opposite metabolic pathways of extracellular nucleotide synthesis and hydrolysis may represent a novel mechanism for regulating an array of cell-specific responses to circulating adenine nucleotides and for terminating their purinergic functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]ATP (spec. act. 5000 Ci/mmol) were
from Amersham (Little Chalfont, U.K.). EC growth factor was from
Boehringer Mannheim GmbH. AB serum was obtained from the Finnish Red
Cross. Organic solvents were from Merck (Rahway, N.J.). Liquid
scintillation mixture Wallac OptiPhase ‘HiSafe’-3 was distributed by
Fisher Co. (Fairlawn, N.J.).TLC plates were Alugram SIL
G/UV254 and Polygram CEL 300 PEI types supplied
by Macherey-Nagel (Duren, Germany). ATP monitoring reagent for
luciferin-luciferase assay was from Bio-Orbit (Helsinki, Finland). All
other reagents were purchased from Sigma.
Cell culture
HUVEC were isolated from fresh human umbilical cords by
treatment with collagenase (type I). The cells were grown in
gelatin-coated tissue culture flasks (Costar, Cambridge, Mass.) at
37°C in a humidified environment containing 5%
CO2. Complete medium contained RPMI 1640
supplemented with 5 U/ml heparin, 4 mM L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, 50 µg/ml EC growth factor, and
10% (v/v) AB serum. HUVEC passages 2–4 were used for experiments. The
integrity and viability of the cells was determined by light microscopy
observations and trypan blue exclusion test.
Measurement of the extracellular metabolism of
3H-labeled adenine nucleotide
Prior to the experiments, HUVEC were seeded onto gelatin-coated
24-well tissue culture plates (Costar) in complete media at a density
of 5 x 104 cells per well. Twenty-four
hours later, confluent HUVEC were rinsed twice with RPMI 1640 and
incubated at 37°C with gentle orbital rotation (60 rpm) in the
starting volume of 0.25 ml RPMI 1640 medium containing 50 µM
3H-labeled ATP, ADP, or AMP as initial
substrates. In certain experiments, unlabeled NTP and NDP were included
into the assay buffer together with [3H]AMP and
[3H]ADP as potential inhibiting and/or
phosphate-donating compounds. Aliquots of the mixture were periodically
applied to an Alugram TLC sheet (
5x104 dpm
per spot), and adenine nucleotides and adenosine were separated by use
of an appropriate solvent system (30)
as described
previously (31)
. Radioactive areas that comigrated with
respective nucleotide/nucleoside standards were scraped into
scintillation vials, extracted from silica with 0.1N HCl, and
quantified by scintillation counting with a Wallac-1409
ß-spectrometer.
To evaluate the extent of adenosine uptake and/or extracellular nucleotide receptor binding, HUVEC were washed twice with RPMI 1640 at the end of appropriate experiments and lysed in 0.25 ml 1 N NaOH containing 0.2% Triton X-100. The radioactivity retained by cells did not exceed 0.5% of the total radioactivity added and this value was not affected by inhibitor of the nucleoside transport dipyridamole (10 µM).
Kinetic analyses of HUVEC ecto-nucleotidases and ecto-nucleotide
kinases
For enzyme kinetic studies, HUVEC were harvested from the tissue
flasks after EDTA treatment (5 mM), washed twice with RPMI 1640, and
incubated in suspension at 37°C. The standard assay contained in a
final volume of 120 µl RPMI 1640 medium, 3–5 x
104 detached cells, 5 mM ß-glycerophosphate,
the indicated concentrations of the corresponding substrate (AMP, ADP,
ATP) with tracer 3H-nucleotide ( 4x105 dpm), and -phosphate-donating ATP or
other NTP as a second substrate (in the case of nucleotide kinase
studies). Incubation times varied from 15 to 60 min, depending on the
substrate concentrations, so that the amount of the converted
3H-nucleotide did not exceed 10% of the
initially introduced substrate. Catalytic reactions were terminated by
applying aliquots of the mixture (2x8 µl) to Alugram TLC sheets, and
3H-labeled substrates and metabolites were
separated and quantified as described above.
Transfer of the -phosphate of [-32P]ATP to AMP
and various NDP
HUVEC were detached as above and incubated at 37°C in 60 µl
RPMI 1640 medium in the presence of 1 µCi
[-32P]ATP as donor of -terminal phosphate
and 1 mM AMP, ADP, GDP, or UDP as phosphate acceptors. After 10 min
incubation, 6 µl of the reaction mixture was spotted onto a
polyethylenimine-cellulose TLC plate. 32P-labeled
ADP and NTP were separated by ascending TLC with 0.75 M
KH2PO4 (pH 3.5), developed
by autoradiography, and quantitated with an imaging analyser.
Quantification of extracellular ATP by luciferin-luciferase assay
Confluent HUVEC growing on 24-well plates were rinsed twice and
incubated at 37°C in 0.5 ml RPMI 1640 medium in the presence of the
indicated concentrations of ADP and/or UTP. Subsamples of the bathing
medium were periodically collected, mixed with EDTA at final
concentration of 5 mM (to prevent further enzymatic ATP hydrolysis)
(32)
, and rapidly centrifuged to remove any detached
cells. The supernatant obtained (40 µl) was pipetted into the wells
of a white nonphosphorescent microplate, placed in a luminometer
(Labsystems Luminoscan 1.2–0) and processed by injection of 150 µl
of ATP monitoring reagent. ATP concentrations were calculated from a
calibration curve constructed at the same time using standard ATP
dissolved in the appropriate solution for each experiment. Because the
commercial ADP preparation contained 0.3% ATP admixture, this
contaminating amount was taken into consideration for calculating ATP
concentration in the experiments with exogenously added ADP.
Data analysis
Data from kinetic experiments were subjected to computer
analysis using the Michaelis-Menten equation to determine the
Km and Vmax values.
In the case of 5'-nucleotidase competitive studies, the
IC50 values were calculated from one-site
competition curves constructed using nonlinear least-squares curve
fitting. All statistical analyses were performed using GraphPad
PrismTM software (version 3.0).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and respective linear formation of the reaction product
[3H]adenosine (Fig. 1B
). Addition of
300 µM unlabeled ATP or ADP to the assay medium significantly
decreased the rate of [3H]AMP hydrolysis to
[3H]adenosine (Fig. 1B
). More
careful TLC analysis of the product formation from metabolism of
[3H]AMP (including its phosphorylated
derivatives) revealed the unexpected clear-cut differences between
these unlabeled nucleotides. Specifically, ATP did not prevent
metabolism of [3H]AMP by HUVEC (Fig. 1A
) but changed its pattern from hydrolytic breakdown to
[3H]adenosine (Fig. 1B
) to further
conversion into 3H-labeled ADP (Fig. 1C
) and ATP (Fig. 1D
). Addition to the assay
medium of 200 µM Ap5A, a specific adenylate
kinase inhibitor (33)
, completely abolished ATP-mediated
[3H]AMP phosphorylation (see Fig. 1
),
suggesting the involvement of adenylate kinase in the measured exchange
activities. In the presence of ADP, most of the
[3H]AMP remained unchanged after 60 min of
incubation with HUVEC (Fig. 1A
), with only a minor fraction
being converted into [3H]ADP (Fig. 1C
). Since ADP-mediated [3H]AMP
conversion to [3H]ADP was inhibited by
Ap5A (data not shown), these results could also
indicate the existence of a HUVEC ecto-adenylate kinase that uses two
ADP molecules for its reverse reaction, followed by involvement of the
reaction product ATP in subsequent transphosphorylation with
[3H]AMP.
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To further elucidate the mechanism of the observed
phosphotransfer reactions, HUVEC were incubated with 300 µM
[3H]AMP and increasing concentrations of
unlabeled ATP or ADP, and formation of the main
3H-labeled AMP derivatives was quantified. Both
ATP and ADP inhibited the rate of [3H]AMP
hydrolysis to [3H]adenosine in a
concentration-dependent manner (Fig. 2A
) with IC50 values of 58 ± 5 and
39 ± 3 µM, respectively. Progressive inhibition of
5'-nucleotidase activity by ATP was accompanied by concomitant
conversion of [3H]AMP into
[3H]ADP (Fig. 2B
) and
[3H]ATP (Fig. 2C
), whereas ADP
induced less pronounced activation of [3H]AMP
phosphorylation. The addition to the assay medium of AMPCP, commonly
accepted as the most powerful inhibitor of membrane-bound
ecto-5'-nucleotidases (1
, 19)
, also caused a progressive
decrease in the rate of [3H]AMP hydrolysis with
an IC50 value of 5.6 ± 0.6 µM (Fig. 2A
); however, no detectable [3H]AMP
phosphorylation was observed in the presence of this nonhydrolyzable
ADP analog (Fig. 2B
, C
). Other naturally occurring guanine,
cytidine and uridine di- and triphosphates also inhibited HUVEC
5'-nucleotidase to a different extent (although not as efficiently as
ATP and ADP) but, as in the case with AMPCP, none of these nucleotides
was capable of inducing any phosphotransfer reactions (data not shown).
Although these studies demonstrate the involvement of highly specific
adenylate kinase in the phosphoryl transfers between
[3H]AMP and ATP, formation of
[3H]ATP as the main end-reaction product
suggests that other kinase activities could contribute to the measured
phosphorylation chain on the HUVEC surface.
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Measurement of ADP-ATP exchange activities mediated by HUVEC
ecto-ATPDase and ecto-NDP kinase
To characterize extracellular ADP-ATP exchange activities,
we applied the same experimental approach: confluent HUVEC were
incubated with 3H-labeled ATP or ADP in the
absence and presence of various NTP as potential donors of -terminal
phosphates. As shown in Fig. 3A
, HUVEC incubation with 50 µM
[3H]ATP as an initial substrate caused its
progressive hydrolysis, with a respective rise in ADP and AMP
concentrations. In this case, the formation of the ultimate reaction
product, adenosine, was significantly delayed with time (Fig. 3A
), presumably due to feed-forward inhibition of
5'-nucleotidase activity by precursor nucleotides (24
, 34)
. This observation concurs with the above competitive data on
the decrease of [3H]AMP hydrolysis in the
presence of ATP and ADP (see Fig. 2A
). HUVEC incubation with
50 µM [3H]ADP also caused its rapid
hydrolysis to the 3H derivatives AMP and
adenosine; surprisingly, there was also a little conversion of
[3H]ADP to [3H]ATP within the first 20 min
of incubation (Fig. 3B
).
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Thus, as expected, product formation from the metabolism of
[3H]ATP (Fig. 3A
) or
[3H]ADP (Fig. 3B
) revealed an
apparent precursor-product reaction, suggesting stepwise ATP and ADP
dephosphorylation via AMP to adenosine. Our previous studies showed
that the initial step of ATP conversion to ADP on the HUVEC surface
exhibits the hallmark characteristics of dedicated E-type ATPases
(8)
, such as Ca/Mg dependence; ineffectiveness of specific
inhibitors of P-, F-, and V-type ATPases; and inhibition by suramin and
other purinergic agents (32)
. Further conversion of ADP to
AMP seems to be mediated by the same endothelial ecto-enzyme that has
been recently identified as ecto-ATPDase (25
, 27
; see
Discussion for details). The existence of a single ATP/ADP-hydrolyzing
enzyme, ATPDase, was considered in additional kinetic experiments (see
below) and its activity was measured using
[3H]ATP or [3H]ADP as
appropriate substrates.
In the presence of 300 µM ATP, the pattern of 50 µM
[3H]ADP metabolism was dramatically changed
(Fig. 3C
) as compared to control HUVEC incubated with
[3H]ADP alone (Fig. 3B
). Concomitant
addition of ATP and [3H]ADP to the cells
induced rapid formation of [3H]ATP and
significantly delayed the rate of appearance of dephosphorylated
3H derivatives, AMP, and especially adenosine.
Contrary to the highly specific adenylate kinase activity, the
conversion of [3H]ADP to
[3H]ATP has broad substrate specificity and can
be induced by ATP, ITP, UTP, and GTP, with the first two nucleotides
being the most active and equipotent (Fig. 4
). No activation of [3H]ATP formation was
detected in the presence of excess inorganic phosphate (Fig. 4)
;
moreover, Ap5A did not exert an inhibitory effect
on the ATP-mediated [3H]ADP conversion to
[3H]ATP (data not shown). These results suggest
the existence of the endothelial ecto-enzyme NDP kinase-mediating
interconversion of extracellular ADP and ATP, whereas neither ATP
synthase nor adenylate kinase contributes to the measured phosphoryl
transfer.
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It should be stressed that use of the confluent HUVEC attached to gelatin-coated plates in the above experiments allowed us to minimize the possible contribution of intracellular pools of purinergic enzymes in the measured exchange activities. Furthermore, no changes in phosphotransfer reactions were observed after pretreatment of the cells with nonspecific P2 antagonist suramin (100 µM) or inhibitor of vesicular transport N-ethylmaleimide (500 µM) (data not shown), thus excluding the alternative possibility of the release of intracellular nucleotide kinases as a consequence of ATP-induced autocrine stimulation of P2 receptors or via other exocytotic mechanisms.
Kinetic analyses of HUVEC ecto-nucleotidases and ecto-nucleotide
kinases
Since ecto-nucleotidases and ecto-nucleotide kinases coexist on
the HUVEC surface and share similarities in substrate specificity, it
is evident that determination of their kinetic parameters could provide
useful information concerning possible relationships between these two
opposite metabolic pathways. The nucleotide-hydrolyzing activities were
determined by HUVEC incubation with increasing amounts of
3H-labeled ATP, ADP, or AMP as initial substrates
(Fig. 5A
). ATPase activity in these kinetic studies was evaluated by
the rate of [3H]ATP degradation to
3H metabolites that were quantified as pooled
ADP, AMP, and adenosine fractions. Likewise, ADPase and 5'-nucleotidase
activities were determined by the rates of formation of
3H derivatives from the hydrolysis of
[3H]ADP and [3H]AMP,
respectively. Statistical analysis of these kinetic data showed that
the ability of HUVEC ATPDase to hydrolyze
[3H]ADP is characterized by 2.2-fold higher
maximal velocity rate (Vmax) and lower
affinity (estimated by the increased apparent Km
value) as compared to [3H]ATP (Table 1
). The obtained ratio of ADP/ATP-hydrolyzing activities is similar to
that described earlier for HUVEC ATPDase (27)
, thus
confirming the high enzyme preference for ADP as substrate. Although
the hydrolysis of [3H]AMP on the HUVEC surface
is characterized by lower Vmax and
Km values compared with
[3H]ADP and [3H]ATP
(Table 1)
, the
Vmax/Km ratios for
all these nucleotide-hydrolyzing activities are comparable and range
within 0.040–0.045.
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Concentration-response curves for nucleotide kinase activities were
generated under conditions that approximated first order rates of
reaction, i.e., one substrate was maintained at a near maximal
saturating concentration whereas the concentration of the second
nucleotide was varied. Kinetic parameters for HUVEC ecto-NDP kinase
were determined in the presence of 800 µM
[3H]ADP as a phosphate acceptor and increasing
amounts of ATP (Fig. 5B
) or other NTP as phosphate donors.
Statistical analysis revealed the preference of ecto-NDP kinase for ATP
and ITP as -phosphate-donating NTP, whereas in the presence of UTP
the rate of [3H]ADP conversion to
[3H]ATP was 50% less (Table 1)
. The
concentration dependence of adenylate kinase activity on unlabeled ATP
was measured in the presence of 500 µM
[3H]AMP (Fig. 5B
). In this case,
however, direct determination of the rate of
[3H]AMP conversion to
[3H]ADP was complicated by further conversion
of the newly formed reaction product to [3H]ATP
through subsequent NDP kinase reaction. Therefore, concentrations of
both 3H derivatives ADP and ATP were pooled and
considered as phosphorylated products formed from
[3H]AMP in the course of adenylate kinase
reaction. Comparison of the kinetic parameters reveals that the
Vmax value for NDP kinase is
10-fold higher than that of adenylate kinase, whereas the
Km values are similar (Table 1)
, suggesting that the
main physiological role of these ecto-nucleotide kinases is the
generation of extracellular ATP as the main end-reaction product.
Direct demonstration of the transfer of -phosphate from ATP to
other nucleotides
Together, the results above demonstrate the existence of reverse
AMP-ADP-ATP exchange activities on the HUVEC surface. Figure 6
represents a pictorial example of these interconversion pathways, where
the formation of high-energy 3H phosphoryl during
HUVEC incubation with [3H]AMP or
[3H]ADP in the presence of unlabeled ATP is
illustrated. It should be taken into account that use of AMP and ADP
with a 3H-labeled adenine ring does not rule out
the probability that these 3H-nucleotides could
be converted to any intermediary derivatives, prior to their
transphosphorylation with ATP.
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Evidence for the existence of ecto-NDP kinase-mediating
phosphotransfers between nucleotides was found by using
[-32P]ATP as donor of phosphoryl groups. As
can be seen in Fig. 7
, HUVEC incubation with 1 µCi [-32P]ATP in
the presence of 1 mM ADP, UDP, or GDP caused a direct transfer of the
-terminal 32P-phosphate from ATP to the
unlabeled NDP with the formation of corresponding
[-32P]NTP. Likewise, the existence of
ecto-adenylate kinase activity was shown with the formation of
32P-labeled ADP under combined treatment of the
cells with [-32P]ATP and AMP (1 mM) and by
blocking such phosphotransfer with the specific enzyme inhibitor
Ap5A (Fig. 7)
. It should be noted that the
addition of [-32P]ATP alone to the control
cells was also accompanied by minor formation of
32P-labeled UTP and GTP. These results indirectly
indicate the existence of low nanomolar concentrations of other,
nonadenine, nucleotides in the vicinity of the HUVEC and concur with
recent data on constitutive release of endogenous UTP along with ATP
from various cell types (4
, 5)
.
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To check whether plasma membrane proteins can be concurrently
phosphorylated under these conditions, the cells were washed at the end
of the appropriate experiments, lysed and subjected to 15% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. No detectable
protein phosphorylation was observed during incubation of the HUVEC
with [-32P]ATP and other nucleotides (data
not shown), suggesting that the major role of these phosphotransfers is
the interconversion of extracellular nucleotides rather than activation
of the surface ecto-protein kinase activities.
Bioluminescent determination of extracellular ATP synthesis
The possibility of ATP synthesis on the HUVEC surface was
independently confirmed by direct measurement of ATP concentration
using a luciferin-luciferase assay and these data can be seen in
Fig. 8
. In the absence of any treatment, the amount of ATP in the medium is
maintained at a low and stable level (14.5±3.8 nM after 30 min
incubation; n=6). Addition of low micromolar concentrations
of exogenous ADP (10–20 µM) caused a progressive increase of
extracellular ATP concentration that was blocked by pretreatment the
cells with adenylate kinase inhibitor Ap5A (5
µM). However, the most pronounced ATP synthesis was observed with the
combined exposure of HUVEC to 10 µM ADP and 50 µM UTP (1.93±0.57
µM after 30 min incubation; n=4), confirming the necessity
of a -phosphate-donating NTP for efficient ADP conversion to ATP.
Likewise, extracellular ATP concentrations reached micromolar level
under combined HUVEC treatment with 10 µM ADP and 50 µM of either
GTP or ITP, whereas the addition of any NTP by itself did not induce
ATP formation, even at concentration ranges of
10-4-10-3 M (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When exogenous 3H-nucleotides were added to the
HUVEC as initial substrates, the levels of metabolites showed their
rapid dephosphorylation through the stepwise reactions: ATP ADP
AMP adenosine. Similar patterns of nucleotide hydrolysis have been
described earlier for different types of endothelial cells (19
, 24
, 34)
and the involvement of three different enzymes in the
observed sequence was proposed (i.e., ecto-ATPase, ecto-ADPase and
5'-nucleotidase). More recent studies suggest that the initial steps of
ATP and ADP hydrolysis are mediated by the same endothelial ecto-enzyme
ATPDase, also identified as surface antigen CD39 (25
26
27
, 35)
. The distinction between the two main members of E-type
ATPase family, ecto-ATPases and ATPDases, is always complicated by
overlapping substrate specificity, coexpression in many tissues, and
close immunological cross-reactivity. In the case of vascular
endothelium, the predominant contribution of a single ATPDase to the
extracellular nucleotide hydrolysis has been confirmed in recent
studies with endothelial cells cultured from ATPDase/CD39-deficient
mice, which were almost completely devoid of both ATP- and
ADP-hydrolyzing activities (22
, 36)
. The last step of AMP
hydrolysis to adenosine on the HUVEC surface can be attributed to the
GPI-anchored ecto-enzyme 5'-nucleotidase, as verified by its
competitive inhibition with a classical inhibitor of membrane-bound
5'-nucleotidases (AMPCP; Fig. 2A
) and its susceptibility to
cleavage by phosphatidylinositol-specific phospholipase C
(32)
.
Although the kinetics of adenine nucleotide breakdown by a cascade of
endothelial ecto-nucleotidases have been extensively characterized and
established as the major effector system for termination of the
purinergic effects of circulating nucleotides (9
, 25)
, the
above data provide additional information concerning accurate kinetic
and competitive parameters for the main nucleotide-hydrolyzing
activities on the HUVEC surface. Furthermore, the important novelty of
this work is the demonstration that this commonly accepted
nucleotide-degrading mechanism prevails only when a particular
nucleotide is present alone in the extracellular milieu. Indeed,
combined treatment of the HUVEC with unlabeled ATP and either
[3H]AMP or [3H]ADP
caused unexpected phosphorylation of
3H-nucleotides via ‘backward’ reactions AMP
ADP ATP, suggesting the coexpression of specific nucleotide kinase
activities on the cell surface.
Competitive and substrate-specificity studies revealed the existence of
an endothelial ecto-NDP kinase that possesses the highest catalytic
activity compared to ecto-nucleotidases and further demonstrated the
expression of another heretofore unrecognized ecto-enzyme, adenylate
kinase, on the cell surface. Although most of these exchange activities
were determined using 3H-labeled nucleotides as
phosphate acceptors, use of [-32P]ATP in
some of our experiments unequivocally confirmed the expression of
adenylate kinase and NDP kinase activities on the HUVEC surface,
mediating direct transfer of 32P-labeled
-phosphate from ATP to AMP or NDP, respectively. The mechanism of
the ecto-enzymatic ADP conversion to ATP was also independently
demonstrated by a luciferin-luciferase assay, where the concentration
of ATP in the bathing medium was dramatically increased under combined
HUVEC exposure to ADP and NTP.
It should be noted that relatively weak but significant ADP conversion
to ATP occurs even in the absence of -phosphate-donating NTP, as
detected by both radio TLC assay and luciferin-luciferase luminometry.
Since the spatial arrangement of adenylate kinase provides a
bidirectional and thermodynamically efficient phosphorelay
(33)
, the equilibrium of forward and reverse reactions in
the presence of excess ADP is shifted toward ATP generation, which
underlies the ADP-ATP exchange activities observed on the cell surface.
In principle, such conversion could also be mediated by another enzyme
ATP synthase that was shown to be expressed on the HUVEC surface
(20)
. However, addition of inorganic phosphate together
with ADP did not induce further activation of ATP synthesis, suggesting
that the observed catalytic reaction was different from that of
conventional ATP synthase.
Adenylate kinase is known to be a highly flexible protein containing
two nucleotide binding domains for ATP and AMP that substantially
change on substrate binding (33)
. Isoforms of adenylate
kinase are implicated in the processing of cellular signals associated
with transfer of high-energy phosphoryl from mitochondria to cellular
ATPases and in regulating ATP-sensitive K+
channel behavior (37
, 38)
, and until now have been only
found in the cytosol, mitochondrial matrix, and mitochondrial
intermembrane space (33
, 37)
. Another enzyme, NDP kinase,
belongs to a large family of structurally and functionally conserved
proteins that are distributed ubiquitously in the nucleus,
mitochondria, cytoplasm, and plasma membranes (14)
. NDP
kinase is a housekeeping enzyme that is essential for DNA and RNA
synthesis; it has been implicated in developmental control, signal
transduction, and tumor metastasis suppression (14
, 39)
.
Although the expression of NDP kinase on the surface of different cell
types has been suggested (5
, 11
, 16
17
18
19)
, only one other
study with human astrocytoma cells has considered the physiological
importance of ecto-NDP kinase activity in defining P2 agonist
activities in nucleotide-mediated signaling (18)
.
Taken together, these findings provide the first kinetic evidence that,
depending on the local nucleotide concentrations, the balanced
metabolism of adenine nucleotides on the cell surface includes both
their hydrolysis and resynthesis. Moreover, we show here that the
interrelation between these two opposite, ATP-consuming and
ATP-generating, metabolic pathways is defined not only by relative
velocities of the involved nucleotide-converting ecto-enzymes, but also
by feed-forward inhibition of 5'-nucleotidase activity by precursor
nucleotides. According to the proposed model, at steady-state
conditions the cells maintain external concentrations of ATP and other
nucleotides at a low and constant level, where hydrolysis is balanced
by a basal (‘constitutive’) release of endogenous nucleotides
(5)
and/or their interconversion via phosphotransfer
reactions (present work). A disturbance of such optimal ratio during
enhanced release of a particular nucleotide to the intercellular space
will result in dominance of stepwise nucleotide degradation to
adenosine. Under these conditions, ATP and its derivatives can be
preferentially delivered to the corresponding substrate binding sites
of cell surface ecto-nucleotidases, as proposed earlier in the
‘two-compartment model’ (24
, 34)
. The joint release of
ATP and either ADP or AMP causes sufficient conformational changes of
corresponding ecto-nucleotide kinases to activate phosphotransfer
reactions and concurrently inhibits ecto-5'-nucleotidase activity,
thereby changing the whole pattern of nucleotide metabolism from
adenosine formation to the continuous resynthesis of high-energy
phosphoryl.
Although the physiological advantages of such exchange activities still
remain to be clarified, we believe that our results provide a
sufficient justification for reexamination of the current concept of
the extracellular nucleotide turnover. These observations are
particularly pertinent for the concept of hemostasis and
thromboregulation, where metabolism of endogenously released ADP/ATP
was shown to be the key component of platelet and vascular endothelial
cell activation responses that culminate in vascular thrombosis
(22
, 27
, 36)
. Identification of phosphotransfers between
adenine and nonadenine nucleotides adds another level of complexity to
understanding the physiological roles of these nucleotides as
extracellular signaling molecules. The release of endogenous guanine
and uridine nucleotides, along with ATP and/or ADP and their
interconversion through ecto-NDP kinase reaction, could represent an
important route for triggering cellular events such as activation of
G-proteins (39)
and signaling via selective purine- and
pyrimidine-specific P2Y receptors (5
, 18)
.
|
ACKNOWLEDGMENTS |
|---|
Received for publication May 4, 2000.
Revision received July 5, 2000.
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REFERENCES |
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