0022-3565/02/3023-992–1001$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 302:992–1001, 2002
Vol. 302, No. 3
33332/1005378
Printed in U.S.A.
Enzyme Kinetics and Pharmacological Characterization of
Nucleotidases Released from the Guinea Pig Isolated Vas
Deferens during Nerve Stimulation: Evidence for a Soluble
Ecto-Nucleoside Triphosphate Diphosphohydrolase-Like
ATPase and a Soluble Ecto-5⬘-Nucleotidase-Like AMPase
SVETLANA T. MIHAYLOVA-TODOROVA, LATCHEZAR D. TODOROV, and DAVID P. WESTFALL
Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada
ABSTRACT
Previously, we have demonstrated that stimulation of the sympathetic nerves of the guinea pig vas deferens evokes release
not only of the cotransmitters ATP and norepinephrine but also
of soluble nucleotidases that break down extracellular ATP,
ADP, and AMP into adenosine. In this study we show that the
apparent Km values of the releasable enzyme activity vary
depending on which of these adenine nucleotides is used as
initial substrate. The Km value for ATP was 33.6 ⫾ 2.3 ␮M,
21.0 ⫾ 2.3 ␮M for ADP, and 10.0 ⫾ 1.1 ␮M for AMP. The ratios
of the Vmax values for each enzyme reaction were 4:2:3. We
have also found a different sensitivity of the metabolism of ATP
and AMP by releasable nucleotidases to known nucleotidase
inhibitors. Suramin inhibited the breakdown of ATP by releasable nucleotidases in a noncompetitive manner and with a Ki
ATP is released as a cotransmitter from cholinergic, adrenergic, and GABAergic neurons (Silinsky et al., 1998; Burnstock, 1999; Jo and Schlichter, 1999). Traditionally, the inactivation of neurotransmitter ATP in both the central and
the peripheral nervous systems has been attributed to its
breakdown by cell membrane-bound enzymes, classified as
ecto-ATPases, ecto-apyrases, and ecto-5⬘-nucleotidases (Zimmermann, 1992; Plesner, 1995).
We have demonstrated, however, that neurogenic stimulaThis work was supported by National Institutes of Health Grant HL-38126
(to D.P.W. and L.D.T.) and the Foundation for Research (to D.P.W. and
S.T.M.T.). A preliminary account of this work has been published as a proceedings article to the Second International Workshop on Ecto-ATPases and
Related Ectonucleotidases, Diepenbeek, Belgium, June 14 –18, 1999.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.033332.
value of 53 ␮M, but had no effect on the breakdown of AMP.
The 5⬘-nucleotidase inhibitor ␣,␤-methylene ADP inhibited the
breakdown of AMP but not that of ATP. Concanavalin A inhibited the breakdown of AMP but had neither inhibitory nor facilitatory effects on the breakdown of ATP. 6-N,N-Diethyl-␤,␥dibromomethylene-D-ATP (ARL67156), an ecto-ATPase
inhibitor, suppressed ATPase and AMPase activities, whereas
NaN3 (10 mM) affected neither reaction, but inhibited the ADP
metabolism. Phosphatase- and phosphodiesterase inhibitors
did not affect the activity of the releasable nucleotidases. This
evidence suggests that the soluble nucleotidases released during neurogenic stimulation of the guinea pig vas deferens combine an ecto-5⬘-nucleotidase-like and an ecto-nucleoside
triphosphate diphosphohydrolase-like activity.
tion of the guinea pig vas deferens dramatically accelerates
the degradation of exogenous ATP (Todorov et al., 1996). The
difference between the rate of degradation of extracellular
ATP by tissue preparations under resting conditions and that
during nerve stimulation appears to be associated with a
release of enzymes that break down ATP as well as ADP and
AMP into ADO (Todorov et al., 1997). Inhibition of the propagation of neuronal action potentials with tetrodotoxin, suppression of adrenergic neurotransmission with guanethidine,
or inhibition of exocytosis by omission of extracellular Ca2⫹
all prevented the release of nucleotidase activity, implying
that sympathetic nerves are the source of the enzyme(s)
(Todorov et al., 1997). Interestingly, the nucleotidase activity
appears to be coreleased with neurotransmitter ATP and not
with the sympathetic cotransmitter NE (MihaylovaTodorova et al., 2001), suggesting that the proteins carrying
ABBREVIATIONS: ADO, adenosine; ENTPDase, ecto-nucleoside triphosphate diphosphohydrolase; ENPPase, ecto-nucleotide pyrophosphatases/phosphodiesterase; GPI, glycosylphosphatidylinositol; EFS, electrical field stimulation; P, sum of products generated in the presence of
superfusate collected under resting conditions; S, sum of products generated in the presence of superfusate collected during nerve stimulation;
HPLC, high-performance liquid chromatography; eATP, 1,N6-etheno ATP; eADP, 1,N6-etheno ADP; eAMP, 1,N6-etheno AMP; eADO, 1,N6-etheno
adenosine; ␣,␤-mADP, ␣, ␤-methylene 5⬘-adenosine diphosphate; IBMX, 3-isobutyl-1-methylxanthine; Con A, concanavalin A.
992
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Received January 22, 2002; accepted May 17, 2002
Characterization of Releasable Nucleotidases
for the releasable nucleotidase activity. Our results suggest
that the sympathetic nerves of the guinea pig vas deferens
release either a soluble, heretofore unidentified enzyme that
combines separate phosphohydrolase and 5⬘-monophosphate-diesterase catalytic activities or a mixture of separate
enzyme entities, including a soluble ENTPDase-like ATPase
and a soluble ecto-5⬘-nucleotidase-like AMPase.
Materials and Methods
Tissue Preparation. Male albino guinea pigs (350 – 400 g) were
killed by decapitation. The vasa deferentia were removed, cleaned of
connective tissue, and the lumen exposed by a section along the
longitudinal axis. Three tissues, each from a different animal, were
loaded in a superfusion chamber (inner volume of 200 ␮l; Brandel
Inc., Gaithersburg, MD). Whatman (Maidstone, UK) 541 filters were
cut to fit both ends of the chamber, which was then inserted vertically into a thermostatic block (36°C) with platinum “screen” electrodes at each end. The tissues were superfused from bottom to top
(2 ml/min) with modified Krebs-HEPES buffer, pH 7.4, of the following composition: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM
CaCl2, 5 mM HEPES, and 11 mM glucose. The buffer was constantly
bubbled with 100% O2.
Nerve Stimulation and Sample Collection Protocols. The
sympathetic nerves of the guinea pig vasa deferentia were stimulated for 30 s by electrical field stimulation (EFS) at 16 Hz, pulse
duration of 0.2 ms, and supramaximal voltage. Three sessions of EFS
were applied to the tissues at 30-min intervals. Samples of the
Krebs-HEPES buffer superfusing the tissue preparations were collected for 30 s before and for 30 s during each of the stimulations in
ice-cold test tubes containing the protease inhibitor leupeptin (1
␮M). The samples were combined in two pools, one designated as
prestimulation or P (collections before stimulation) and the other
designated as S (collections during stimulation). If not used the same
day, the pooled samples were frozen in liquid nitrogen and then
stored at ⫺86°C.
HPLC-Based Assay for Nucleotidase Activity. To study the
properties of the releasable neuronal nucleotidases, the fluorescent
1,N6-etheno analogs of adenine nucleotides were used as substrates
(Fig. 1). The rationale of the assay is based on the fact that the
sequential dephosphorylation of 1,N6-etheno ATP (eATP) results in
formation of 1,N6-etheno ADP (eADP) followed by formation of 1,N6etheno AMP (eAMP), and finally formation of 1,N6-etheno adenosine
(eADO). Each of the substrates and the resulting metabolites were
quantified after separation by HPLC coupled with fluorescent detection. With this method the rate of hydrolysis of the substrate, as well
as the type and rate of generation of the products were evaluated in
a single chromatogram. eATP was used as initial substrate to study
ATPase activity (Fig. 1A), whereas eADP was used to study ADPase
activity (Fig. 1B), and eAMP was used to study AMPase activity (Fig.
1C)
HPLC-Based Assays for Adenine Nucleotides and ADO.
ATP, ADP, AMP, and ADO were analyzed as described previously
(Todorov et al., 1996). The etheno-adenine purines were separated on
a gradient HPLC system equipped with a Resolve radial pack cartridge (8NV Ph 4 ␮m; 8 ⫻ 10 mm) (Waters, Milford, MA). The
amount of each adenine purine was quantified using an RF 535
fluorescent monitor (Shimadzu, Columbia, MD) at an excitation
wavelength of 230 nm and an emission wavelength of 420 nm. Buffer
solutions consisted of 0.1 M phosphate (KH2PO4, pH 6.0) (buffer A)
and 75% 0.1 M phosphate and 25% methanol (buffer B). The adenine
nucleotides and ADO were separated using a gradient in which the
concentration of buffer B was increased from 0 to 100% in 8 min
according to Waters gradient profile 7. The HPLC equipment was
controlled by, and data collected by, a Pentium II computer equipped
with an LAC/E card and Millenium 2010 Chromatography Manager
software (Waters). Identification of individual peaks in chromato-
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the enzyme activity originate from a putative “ATP storage
vesicle” rather than from a catecholamine storage vesicle.
We investigated the possibility that known ATPases, activated during the process of exocytosis, may be involved as
releasable ATPases. The vacuolar H⫹-transporting ATPase,
the Na⫹/K⫹-ATPase, the multidrug-resistance channel, and
the cytosolic N-ethylmaleimide-sensitive fusion protein were
rejected as possible candidates based on the evidence that
their specific antagonists did not inhibit the releasable ATPase activity. We have found, however, that suramin and
6-N,N-diethyl-␤,␥-dibromomethylene-D-ATP
(ARL67156)
were potent inhibitors of the releasable ATPase activity
(Todorov et al., 1997). Because both drugs have been shown
to inhibit ecto-ATPases (Crack et al., 1995) we speculated
that the releasable nucleotidases might share structural similarities with ecto-ATPases (Kennedy et al., 1997; Todorov et
al., 1997).
Interestingly, none of the known members of the mammalian ecto-ATPase gene family, recently renamed ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) (EC
3.6.1.5) (Zimmermann and Braun, 1999), matches the neuronally released enzymes with regard to both substrate specificity and membrane localization. In fact, the ENTPDase
members that possess ATPase activity such as ENTPDase1
(Maliszewski et al., 1994; Kaczmarek et al., 1996), ENTPDase2 (Kegel et al., 1997; Mateo et al., 1999; Vlajkovic et al.,
1999), or ENTPDase3 (Smith and Kirley 1998) are membrane-bound proteins, whereas expression of the potentially
soluble members such as the ENTPDase5 or ENTPDase6
(Chadwick and Frischauf, 1998) revealed that they poorly
metabolize ATP (Mulero et al., 1999; Braun et al., 2000;
Hicks-Berger et al., 2000). Moreover, none of the members of
the ENTPDase family hydrolyze nucleotide monophosphates
to nucleosides. Because soluble ENTPDases with ATPase
activity have not as yet been identified, and the enzyme(s)
released upon stimulation of the sympathetic nerves of the
guinea pig vas deferens hydrolyzes AMP as well as ATP and
ADP, other potential candidates for the releasable nucleotidases should be considered.
Members of the mammalian family of ecto-nucleotide pyrophosphatases/phosphodiesterases (ENPPases) break down
nucleotide triphosphates, diphosphates, and dinucleotidepolyphosphates into monophosphates. Although ENPPases
have a short membrane-spanning domain, the large extracellular domain containing the catalytic center could be
cleaved from the membrane and released in soluble form (for
review, see Bollen et al., 2000). However, ENPPases do not
metabolize AMP to adenosine.
Alkaline- and tissue-nonspecific acid phosphatases are
able to hydrolyze ATP, ADP, and AMP and exist in both
glycosylphosphatidylinositol (GPI)-anchored and soluble
forms (Ohkubo et al., 2000). Ecto-5⬘-nucleotidase (E.C.
3.1.3.5) or CD73 (Resta et al., 1993) is a 5⬘-monophosphoadenosine- or inosine-specific phosphatase (Zimmermann,
1992). Upon activation of phosphatidylinositol-specific phospholipase C, the GPI-anchored ecto-protein could be released
in soluble form, while retaining its catalytic activity (Misumi
et al., 1990; Lehto and Sharom, 1998).
Given the broad, mono-, di-, or triphosphonucleotide specificity of the releasable neuronal nucleotidases we explored
the possibility that ENTPDases, ENPPases, phosphodiesterases, 5⬘-nucleotidases, or phosphatases may be responsible
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grams was by comparison with the retention times of known ethenoadenine purine standards, and the concentration was determined by
peak area per picomole relationship compared with standards. Standards were run with each set of samples.
Preparation of 1,N6-Etheno Derivatives of ATP, ADP, AMP,
and ADO. Stock solutions of eATP, eADP, and eAMP were prepared
by incubation of ATP, ADP, and AMP (1.5 ⫻ 10⫺2 mol/l) in citric
phosphate buffer, pH 4, in the presence of 2-Cl-acetaldehyde for 40
min at 80°C. Furthermore, serial dilutions (3 ␮M–10 mM) of the
stock solutions were prepared using deionized water (18 M⍀) and
stored at ⫺20°C. The etheno-derivatives of adenine nucleotides are
stable at ⫺20°C for years and at room temperature for several days.
Hydrolysis of ATP, ADP, and AMP and Their Etheno-Derivatives by Commercially Available Enzymes. To test whether the
modification of the molecules of ATP, ADP, or AMP by the ethenogroup addition affects the enzymatic degradation of adenine nucleotides we have compared the rate of breakdown of native ATP and
ADP with the rate of breakdown of their etheno-derivatives (eATP
and eADP) by apyrase VI and VII purchased from Sigma-Aldrich (St.
Louis, MO). The hydrolysis of AMP and that of eAMP by 5⬘-nucleotidase from Crotalus atrox venom (Sigma-Aldrich) was also evaluated. The etheno-analogs were metabolized by the commercially
obtained enzymes with rates closely comparable with the rates for
the nonmodified substrates (data not shown).
Time Course of Product Formation. eATP, eADP, or eAMP (1,
10, and 100 ␮M) was incubated with superfusate S or P for 0, 0.5, 1,
2, and 4 h at 37°C. At the end of the incubation periods the reactions
were stopped with acidification with ice-cold citrate phosphate
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Fig. 1. Chromatographic image of etheno-purines before (Aa, Ba, and Ca) and after (Ab, Bb, and Cb) exposure to releasable nucleotidases. Structural
formulae and representative chromatograms of eATP (Aa), eADP (Ba), and eAMP (Ca) in Krebs-HEPES buffer. The structural formula of eADO is
shown as an inset in Cb. The etheno-modification, consisting of insertion of acyl group between nitrogen 1 and 6 of the adenine moiety, renders the
molecule fluorescent but leaves unaltered the phosphate chain. Note that an invariable with time nonenzymatic dephosphorylation product (less than
5% for eATP, less than 8% for eADP, and less than 2% for eAMP) is always detected in the solutions. eATP incubated for 60 min at 37°C with
superfusate S collected during nerve stimulation of the guinea pig isolated vas deferens was metabolized to eADP, eAMP, and eADO (Ab). Likewise,
in presence of superfusate S, eADP was metabolized to eAMP and eADO (Bb), whereas eAMP was broken down to eADO (Cb). Metabolism of
etheno-adenine nucleosides did not occur in presence of superfusate P, collected before the onset of the nerve stimulation of the guinea pig isolated
vas deferens (data not shown).
Characterization of Releasable Nucleotidases
Statistical Analysis. Data were evaluated using GraphPad
Prism software, version 3. Means were compared using standard t
test, and p ⬍ 0.05 was considered to indicate a statistically significant difference.
Results
Substrate Specificity of Releasable Nucleotidases.
eATP, eADP, and eAMP were all metabolized in the presence
of superfusate from nerve-stimulated guinea pig vas deferens
tissue preparations (S) (Fig. 1b) but remained unaffected by
superfusate collected under resting conditions (P) (data not
shown). The hydrolysis was time-dependent, sequential, and
unidirectional from ATP to ADO. eADP was the first product
formed from eATP, whereas eAMP and eADO appeared later
in the time course of the reaction. When eADP was used as
substrate, eAMP appeared first, and eADO was formed later
(Fig. 1Bb). E-ADO was the first and only product during the
time course of the metabolism of AMP (Fig. 1Cb). If sufficient
time was allowed, eADO was the end product of the reaction
regardless of whether eATP, eADP, or eAMP was used as
initial substrate.
Increase in Enzyme Activity after Sample Concentration. The volume of samples of superfusate was reduced
by filtration through Centricon filters with a membrane pore
size cutoff of 30 kDa. The ATPase, ADPase, and AMPase
activities were retained above the filter. There was no enzyme activity in the filtrate. In addition, we have observed an
increase in specific activity (determined as activity per microliter of superfusate) consistent with concentration of enzyme(s) with size larger than 30 kDa. Dilution of the superfusate, on the other hand, led to a proportional decrease in
specific enzyme activity.
Kinetic Constants of ATPase, ADPase, and AMPase
Activity. The Michaelis-Menten plots of substrate concentration versus product velocity for all three activities and the
Lineweaver-Burk reciprocal plots are shown in Fig. 2. The
product velocity increased with the increase of the substrate
concentrations (0.3–300 ␮M) according to a rectangular hyperbola. The Km value for the ATPase activity, calculated
from 28 separate experiments, was 33.6 ⫾ 3.2 ␮M and the
Vmax was 0.204 ⫾ 0.003 pmol/min/␮l of superfusate (inserted
table in Fig. 2). The ADPase activity had a Km value of 21.0 ⫾
2.3 ␮M and Vmax value of 0.111 ⫾ 0.002 pmol/min/␮l of
superfusate (n ⫽ 4). The Km value of the AMPase activity was
10.0 ⫾ 1.1 ␮M, and the maximal velocity was 0.168 ⫾ 0.003
pmol/min/␮l of superfusate (n ⫽ 12). These data suggest that
the AMPase operates at a slightly slower rate than the ATPase. The ADPase was the slowest of the three, having only
one-half and two-thirds of the maximal velocities of the ATPase and the AMPase, respectively. The Vmax/Km ratio revealed that the AMPase had a three-fold higher efficiency
than the ATPase or the ADPase. The ratio of the maximal
velocities of the ATPase/ADPase was 2:1. ATP is therefore
preferred 2-fold over ADP, which is consistent with the possibility that a single ENTPDase-like enzyme is responsible
for the metabolism of both ATP and ADP (Plesner, 1995).
Calcium and Magnesium Dependence of Nucleotidase Activity. Previously, we have demonstrated that omission of Ca2⫹ from the superfusing solution or addition of
cadmium (Cd2⫹) abolishes both the nerve stimulation-evoked
release of neurotransmitters and the release of nucleotidase
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buffer, pH 4, and the samples analyzed for ATP, ADP, AMP, and
ADO by HPLC.
Increase in Enzyme Activity with Protein Concentration.
Samples of superfusate diluted 2-, 4-, or 8-fold with Krebs-HEPES
buffer were tested for ATPase and AMPase activity. Concentration of
enzyme(s) was achieved by reduction of the volume of the superfusate by filtration through Centricon centrifugal filters (Millipore
Corporation, Bedford, MA) with membrane pore size cutoff of 30
kDa. Typically, a 2-ml sample was reduced to 40 ␮l. Enzyme activity
was tested using 5, 10, and 20 ␮l of the concentrated sample, corresponding to theoretical 12.5-, 25-, and 50-fold concentration of proteins with molecular size higher than 30 kDa.
Kinetic Constants of the Releasable Nucleotidase Activity.
Under the standard protocol used to study the enzyme activity of
releasable nucleotidase, 5 ␮l of stock solution of eATP, eADP, or
eAMP at a given concentration (3 ␮M–10 mM) was added to a
mixture of 20 ␮l of superfusate collected during neurogenic stimulation of the guinea pig vas deferens (source of enzyme activity) and 25
␮l of Krebs-HEPES buffer, pH 7.4. A similar sample was run using
superfusate P. After incubation for 60 min at 37°C, the reaction was
stopped by addition of 100 ␮l of ice-cold citrate phosphate buffer, pH
4. Aliquots (100 ␮l) were injected into the HPLC system for evaluation of the adenine nucleotides and adenosine present in the sample.
The enzyme activity was estimated from the difference between the
sum of products generated in the presence of superfusate collected
during nerve stimulation (S) and the sum of products generated in
the presence of superfusate collected under resting conditions (P).
This difference (net product) was expressed in picomoles per minute
per microliter of superfusate (pmol/min/␮l). Normalization by microliters of superfusate was used to replace the conventionally used
normalization by protein weight. Kinetic constants (Km and Vmax)
were derived from Michaelis-Menten plots using the nonlinear regression analysis of GraphPad Prism software, version 3 (GraphPad
Software, San Diego, CA).
Calcium and Magnesium Dependence of Releasable Nucleotidases. Nucleotidases were released in Krebs-HEPES buffer as
described above. The collected samples were desalted by centrifugation through a Centricon filter, with a membrane pore cutoff of 30
kDa and then restored to their initial volume with Ca2⫹- and Mg2⫹free Krebs-HEPES buffer. Because the initial concentration of Ca2⫹
and Mg2⫹ in the superfusing buffer was 1.5 and 1 mM, respectively,
the concentration of the cations in the restored samples was estimated to be less than 0.075 and 0.05 mM, respectively. ATPase and
AMPase activities were tested under these low Ca2⫹ and Mg2⫹
conditions as well as after supplementation with Ca2⫹ or Mg2⫹ to
final concentrations of 1, 2, 3, and 10 mM. ATPase and AMPase
activities were also tested in the presence of 5 mM EGTA at pH 7.4.
In another set of experiments, vasa deferentia were stimulated to
release nucleotidases while superfused with buffer from which Mg2⫹
was omitted. The superfusate was tested for ATPase and AMPase
activities under these low-Mg2⫹ conditions as well as after Mg2⫹ was
added to achieve final concentrations of 0.6, 1.2, 2.4, 4.8, and 9.6 mM.
Chemicals. The following chemicals were purchased from SigmaAldrich: adenosine 5⬘-triphosphate (disodium salt); adenosine 5⬘diphosphate (sodium salt); adenosine 5⬘-monophosphate (sodium
salt); adenosine (hemisulfate salt); ␣,␤-methylene adenosine 5⬘diphosphate (␣,␤-mADP); chemiacetal; citric acid; concanavalin A;
EGTA; HEPES; levamisole; NaN3; Na2PO4; phosphates inhibitor
cocktail II; KH2PO4; apyrase VI and VII; 5⬘-nucleotidase from C.
atrox venum; and 3-isobutyl-1-methylxanthine (IBMX), p-nitrophenyl 5⬘-thymidine monophosphate. Suramin hexasodium ([8-(3-benzamido-4-methylbenzamido) naphthalene-1,3,5-trisulphonic acid])
and ARL67156 were purchased from Sigma/RBI (Natick, MA). Methanol was purchased from B & J (Muskegon, MI). 2-Cl-acetaldehyde
was prepared in our laboratory as described previously (Todorov et
al., 1996). The Centricon centrifugal filter device (model 30; Amicon
Bioseparations) was purchased from Millipore Corporation.
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activity from the guinea pig vas deferens. These data have
suggested that neuronal nucleotidases are released by a calcium-dependent mechanism (Todorov et al., 1997). To support the release of enzymes we had to maintain calcium in
the superfusing solution. Magnesium, however, was not required for neuronal release of ATP, norepinephrine, or soluble enzymes. We therefore carried out release experiments
using buffer containing calcium (1.5 mM) but not magnesium
ions. The enzyme activity of these samples increased following a rectangular hyperbola when Mg2⫹ was increased from
0.6 to 4.8 mM. Maximal increase of the ADPase activity
(12%), ATPase activity (8%), and AMPase activity (4%) was
achieved when the Mg2⫹ concentration was increased to 2.4
mM. Further increase of Mg2⫹ concentration to 9.6 mM led to
a decline in enzyme activity. Half-maximal activation of the
ATPase, ADPase, and AMPase was achieved with 0.7, 1.3,
and 0.85 mM Mg2⫹, respectively.
Treatment of samples of superfusate S with EGTA (2 mM,
pH 7.4) abolished the ATPase, ADPase, and AMPase activities. Samples of superfusate S desalted by reduction of the
fluid volume and restored back to the initial volume with
Ca2⫹- and Mg2⫹-free buffer showed a 75% decrease of their
initial activities. When supplemented with Ca2⫹ or Mg2⫹ the
enzyme activities of the samples of superfusate increased
according to a rectangular hyperbola. Half-maximal activation of the ATPase was achieved with 2.6 ⫾ 0.2 mM Ca2⫹ and
with 2.4 ⫾ 0.1 mM Mg2⫹. Half-maximal activation of the
AMPase was achieved with 2.5 ⫾ 0.3 mM Ca2⫹ and with
2.3 ⫾ 0.2 mM Mg2⫹. These data demonstrate that each of
these cations is sufficient to fully activate the ATPase or
AMPase activities of the releasable nucleotidase.
Pharmacological Characteristics of Releasable Neuronal Nucleotidases. The effects of ecto-ATPase inhibitors
on the ATPase and AMPase activities are shown in Fig. 3.
Suramin inhibited the releasable ATPase activity in a dosedependent and noncompetitive manner (Fig. 3A) with a Ki
value of 53 ␮M, derived from Dixon plot. On the other hand,
suramin had no effect on the AMPase activity (Fig. 3B).
ARL67156, previously known as FPL67156, is an ATP
analog devoid of purinergic receptor activity that has been
promoted as a selective ecto-ATPases inhibitor with a Ki
value of 5.2 (Crack et al., 1995). In addition, it has been
shown that ARL67156 increases the ATP-induced contractile
responses of smooth muscle tissue preparations, presumably
by protecting extracellular ATP from degradation by ectoATPases (Westfall et al., 1996). We tested the ability of
ARL67156 to inhibit the breakdown of eATP (Fig. 3C) and
eAMP (Fig. 3D) by releasable nucleotidases. ARL67156 inhibited ATPase activity in a noncompetitive manner with a
concentration-dependent linear decrease of Vmax(app) and a
linear increase of Km(app). The Ki, calculated from replots of
the slope [Km(app)/Vmax(app)], derived from reciprocal transforms versus inhibitor concentrations (Plowman, 1972), was
55.8 ␮M (Fig. 3E). ARL67156 also inhibited the rate of the
AMPase activity (Fig. 3D). The mode of this inhibition was
complex and seemed consistent with the possibility that
ARL67156 binds to more than one enzyme or more than one
binding site. Analysis of the changes in Vmax(app) with increase in inhibitor concentrations revealed that the maximal
decrease in Vmax (about 30%) occurred when ARL67156 concentration was increased from 0.1 to 3 ␮M. Further increase
(10 –100 ␮M) had less effect on Vmax. There was also an
increase in Km(app) parallel to the increase in concentration of
this inhibitor. Within the range from 0.1 to 3 ␮M the Km
value almost doubled. With further increase of inhibitor (10 –
100 ␮M), Km(app) continued to increase, whereas Vmax remained constant. It seems possible, therefore, that
ARL67156 binds to a “high-affinity” binding site, leading to a
noncompetitive inhibition of the AMPase activity. This binding site appears to saturate at low micromolar inhibitor concentrations. At higher concentrations ARL67156 acts as a
competitive inhibitor, because Vmax value does not change,
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Fig. 2. Michaelis-Menten plots and kinetic constants (Km and Vmax) for the
neuronal ATPase, ADPase, and AMPase released from the guinea pig vas
deferens during nerve stimulation. Increasing concentrations of e-ATP, eADP, or e-AMP were used as substrates for the ATPase, ADPase, and
AMPase, respectively. The relationships between product velocity (net
product) and substrate concentrations
fit a rectangular hyperbola for each of
the substrates (A). B, LineweaverBurk reciprocal plots of the ATPase
(eATP used as substrate), ADPase
(eADP used as substrate), and the
AMPase (eAMP used as substrate) activities. The Km and Vmax values for
each of the enzyme reactions are presented in the table. Note that the ratio
of maximal velocities of ATPase/ADPase/AMPase approximates 4:2:3.
Characterization of Releasable Nucleotidases
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Fig. 3. Inhibitory profiles of suramin (A and B) and ARL67156 (C and D) on releasable ATPase (A, C, and E) and AMPase (B, D, and F) activities.
Guinea pig vasa deferentia were stimulated for 30 s with EFS (16 Hz, 0.2 s, 80 V) and samples of the superfusing solution were collected before (P)
and during stimulation (S). Samples of superfusate (20 ␮l) in presence of increasing concentrations (0.3–1000 ␮M) of eATP in a total incubation volume
of 50 ␮l were incubated at 37°C for 60 min. At the end of the incubation period the reaction was stopped with addition of citric phosphate buffer, pH
4, and the amount of metabolites formed (i.e., eADP, eAMP, and eADO) was analyzed using HPLC coupled with fluorescent detection. The releasable
neuronal activity was estimated as the ATPase activity present in superfusate collected during EFS from which the resting activity (activity in sample
P) has been subtracted. The velocity of the ATPase reaction was expressed as the amount of product [eADP ⫹ eAMP ⫹ eADO (in pmol)] formed per
minute per microliter of superfusate. Releasable AMPase activity was tested in a similar way using eAMP as substrate and the velocity of the AMPase
reaction was evaluated as the amount of eADO formed per minute per microliter of superfusate. Inhibitors were added to the reaction mixture 15 min
before addition of substrate. In A, substrate (eATP) versus velocity plot of releasable ATPase in absence and in presence of varying concentrations of
suramin (1–100 ␮M). Suramin inhibits ATPase activity in a noncompetitive manner. As shown in B, suramin has no effect on releasable AMPase
activity. ARL67156 inhibited releasable ATPase activity in a noncompetitive manner (C). In E, slope versus inhibitor replots representing the changes
of the apparent Km/Vmax ratio of the ATPase reaction as function of increasing ARL67156 concentrations demonstrate a Ki value of 55.8 ␮M. In D,
complex inhibition of AMPase activity by ARL67156 consistent with binding of inhibitor to more than one enzyme binding sites in a noncompetitive
way below 3 ␮M and in a competitive manner above 3 ␮M. In F, slope versus inhibitor concentration replots, demonstrating a Ki(1) value of 1.7 ␮M
for the high-affinity binding site and a Ki(2) value of 75 ␮M for the low-affinity binding site of ARL67156 to the releasable AMPase.
whereas Km value increases. Slopes of the reciprocal curves
within the above-defined inhibitor ranges were linear. The
abscissa intercept of the replots was used to determine the Ki
value for the high-affinity binding site [Ki(1) ⫽ 1.7 ␮M] and
for the “low-affinity” binding site [Ki(2) ⫽ 75 ␮M] (Fig. 3F).
The effects of NaN3 (5 and 10 mM) on the kinetic constants
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Mihaylova-Todorova et al.
of the releasable ATPase and the releasable AMPase are
presented in Fig. 4, A and B, respectively. Sodium azide had
no effect on the rate of hydrolysis of ATP and AMP by the
releasable enzymes. However, when eADP (100 ␮M) was
used as substrate, a 30% inhibition of the ADP hydrolysis in
presence of 10 mm NaN3 was observed (data not shown).
We tested the effect of ␣,␤-mADP, an ecto-5⬘-nucleotidase
antagonist (Knofel and Strater, 2001), on the releasable nucleotidase activities. ␣,␤-mADP inhibited the releasable AMPase activity in a competitive manner (Fig. 4D) with a hyperbolic increase in Km(app) value and no change in Vmax
value as inhibitor increased. The concentration of inhibitor
that doubled the Km value of the AMPase reaction was esti-
mated to be 0.0125 ␮M. However, ␣,␤-mADP failed to produce any inhibitory effect on the ATPase activity (Fig. 4C).
Levamisole (10 mM) (Fig. 4, E and F) and phosphatase
inhibitor cocktail II (data not shown), inhibitors of phosphatases, and IBMX (10, 100, and 1000 ␮M), a phosphodiesterase inhibitor, did not affect either releasable ATPase or AMPase activity (data not shown).
We tested the effect of para-nitrophenyl tymidine 5⬘-monophosphate (100 ␮M), a specific substrate for ecto-nucleotide
pyrophosphatases/phosphodiesterases, on the releasable ATPase activity using eATP (100 ␮M) as substrate. para-Nitrophenyl tymidine 5⬘-monophosphate did not affect the metabolism of eATP, suggesting that it does not compete for the
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Fig. 4. Inhibitory profile of sodium azide, ␣,␤-mADP, and levamisole on releasable ATPase (A, C, and E) and AMPase (B, D, and F) activity.
Superfusate was collected and enzyme reactions were carried out as described in Fig. 3. Sodium azide (5 and 10 mM) does not affect the releasable
ATPase (A) or AMPase (B) activities. ␣,␤-mADP (0.1, 1, and 10 ␮M), a specific inhibitor of ecto-5⬘-nucleotidase inhibited competitively releasable
AMPase activity (D), but had no effect on releasable ATPase activity (C). Levamisole (10 mM), a noncompetitive inhibitor of alkaline phosphatase, had
no effect on releasable ATPase (E) or releasable AMPase (F).
Characterization of Releasable Nucleotidases
ATP binding site of the releasable enzyme. It seems, therefore, that ENPPases do not contribute for the releasable
ATPase activity.
It has been shown that the mannose-binding lectin Con A
inhibits the activity of 5⬘-nucleotidases by a noncompetitive
mechanism (Zimmermann, 1992). In our experiments Con A,
at 0.1 ␮M, completely abolished the releasable AMPase activity. However, Con A (0.1, 0.5, and 1 ␮M) had neither
inhibitory nor stimulatory effects on the releasable neuronal
ATPase activity.
Discussion
complex of the ENDPase1 exists in a single form, whereas
the metal-ADP-enzyme complex has two states, one corresponding to the intermediate complex formed during ATP
hydrolysis and the second corresponding to the ADP binding
as substrate for further hydrolysis.
Knowles and Nagy (1999) have shown a nucleotide-substrate-dependent inhibition effect of sodium azide on the
chicken oviduct ecto-ATPDase. The effect of azide was prominent on the ADP hydrolysis, whereas the ATP hydrolysis
was less influenced. Herein, we show that the soluble ATPase
activity was not affected by sodium azide, but the rate of the
ADPase activity was decreased. This is consistent with the
hypothesis that the enzymes released upon nerve stimulation of the guinea pig vas deferens include an ATPDase.
The Km value of the releasable ATPase (33 ␮M) determined
in this work seems similar to the Km values of ecto-ATPases
purified from synaptosomes of the rat and mouse cortex (Km
⫽ 39 –53 ␮M; Nagy et al., 1986), but was lower than that of
ATPase from rat striatal cholinergic synapse (Km ⫽ 131 ␮M;
James and Richardson, 1993). The expressed recombinant
ENTPDases, however, show Km values for ATP ranging from
75 to 400 ␮M that are in general higher than the ones found
in tissues. ENTPDase1 and 3 use ADP as well as ATP as
substrates, whereas ENTPDase2 is mostly an ATPase because it prefers ATP 10 times more than it prefers ADP.
ENTDPase1 metabolizes ATP directly to AMP, and ADP does
not appear as free product in the reaction. The guinea pig vas
deferens neuronal nucleotidases generate ADP as a free
product resulting from the ATP hydrolysis. ADP is detected
in the reaction before it is hydrolyzed further to AMP. The
velocity of ATP hydrolysis is 2-fold higher than that of ADP.
The soluble ATPase released from the sympathetic nerves of
the guinea pig vas deferens is therefore a triphosphatediphosphohydrolase-like enzyme. Because ADP accumulates
as a product of the metabolism of ATP, it seems that the
releasable ATPase is more similar to ENTPDase3, which
prefers ATP 3 to 4 times more than ADP (Smith and Kirley,
1998) than it is to ENTPDase1, which does not discriminate
between ATP and ADP (Wang and Guidotti, 1996). On the
other hand, recent work of Chen and Guidotti (2001a) demonstrated that the velocity of the ATP hydrolysis and the
preference for ADP binding are functions of the oligomerization state of the ENTPDase monomers. Disruption of the
tetrameric organization of the membrane-bound rat ENTPDase1 by either detergent solubilization or truncation of the
N- and C-terminal segments of the recombinant protein decreased its preference for ADP 3.5 times and ADP appeared
in solution during the metabolism of ATP. The Km value for
ATP was also decreased into the low micromolar range. It
seems, therefore that the kinetics of the releasable ATPase
from the guinea pig vas deferens reported in the current work
approximates the kinetics of the C-terminal- and N-terminaltruncated rat ENTPDases. This opens the possibility that the
releasable ATPases could in fact represent a proteolytic
cleavage form of the membrane-bound ENTPDases.
The activity of ecto-ATPase in neuronal (Marti et al., 1996)
and non-neuronal tissues (Bultmann et al., 1996) is inhibited
by suramin. Suramin has also been shown to inhibit the
ATPase activity of the Chinese hamster ovary cells transfected with the rat brain ecto-ATPase gene, but not that of
Chinese hamster ovary cells transfected with the ectoapyrase gene (Heine et al., 1999). Herein, we show that the
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The results reported herein confirm our previous findings
that the breakdown of extracellular ATP and consequently
the level of extracellular ADO are regulated by soluble nucleotidases, released upon nerve stimulation of the sympathetic
nerves of the guinea pig vas deferens (Todorov et al., 1996,
1997; Mihaylova-Todorova et al., 2001). Concentration of superfusate collected during nerve stimulation leads to an increase in nucleotidase activity and suggests that soluble proteins with a size greater than 30 kDa are involved.
In an attempt to more completely understand the nature of
the releasable nucleotidases, we have examined the effects of
several pharmacological agents that are known to inhibit
ecto-phosphatases and ecto-phosphodiesterases. These families of adenine nucleotide-metabolizing enzymes exhibit
broad substrate specificity. There is also evidence that ectophosphatases may be released from the cell membrane upon
activation of endogenous phospholipases and cleavage of the
anchoring GPI (Hooper, 1997). We have found, however, that
the activity of releasable nucleotidases is not affected by
either levamisole, a specific alkaline phosphatase antagonist,
or by the phosphatase inhibitor cocktail II (Sigma-Aldrich),
which is designed to block the actions of a wide range of
phosphatases. These results suggest that alkaline-, acid-,
neutral-, or protein-tyrosine phosphatases are not involved
in the nerve stimulation-triggered metabolism of adenine
nucleotides in the guinea pig vas deferens. Likewise, IBMX,
a nonselective phosphodiesterase antagonist, failed to inhibit
the ATPase and AMPase activities, thereby excluding the
possibility that releasable nucleotidases may share catalytic
properties with ecto-phosphodiesterases. Additionally, paranitrophenyl thimidine monophosphate, a preferred substrate
of ENPPases, had no influence on the eATP metabolism by
releasable nucleotidases, suggesting that ENPPases do not
contribute to the soluble ATPase activity.
Several lines of evidence presented in this study support
our previous hypothesis that releasable nucleotidases may
share catalytic properties with ecto-ATPases (Kennedy et al.,
1997; Todorov et al., 1997; Westfall et al., 2000b). Our results
show that like most ecto-ATPases the releasable nucleotidases depend on either Ca2⫹ or Mg2⫹ for activity. Removal of
divalent cations by chelation or buffer exchange abolishes the
ATPase, ADPase, and AMPase activities. Addition of Ca2⫹ or
Mg2⫹, on the other hand, restored these activities. The effects of Ca2⫹ and Mg2⫹ were additive, suggesting that both
cations use the same mechanism of activation. Mg2⫹ activated ADPase more than it activated ATPase or AMPase,
suggesting that small differences in the metal ion coordination for ATP and ADP may exist. Recently, Chen and
Guidotti (2001b) demonstrated that the metal-ATP enzyme
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Mihaylova-Todorova et al.
noted, however, that this enzyme does not metabolize AMP.
If the releasable nucleotidase described herein is a single
enzyme then it should have at least two separate active
centers that may be independently modulated by ecto-ATPase- and ecto-5⬘-nucleotidase inhibitors.
Acknowledgments
We are grateful to Professor William Welch (Department of Biochemistry, University of Nevada School of Medicine) for advice on
the enzyme kinetic studies and to Professor William Fleming for
comments on the manuscript.
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