Effects of cooling and ARL 67156 on synaptic ecto-ATPase activity in
guinea pig and mouse vas deferens
Para Ghildyal, Rohit Manchanda*
Biomedical Engineering Group, School of Bioscience; Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, Maharashtra 400076, India
Abstract
We have studied the influence of temperature and ARL 67156 on ATP hydrolysis in mouse and guinea pig vas deferens in order to explore
the properties of the enzymatic inactivation mechanism proposed to regulate purinergic neurotransmission at the sympathetic neuromuscular
junction of smooth muscle. The ectonucleotidase activity was determined by using the malachite green method to measure the inorganic
phosphate (Pi) liberated with ATP used as a substrate. ATP hydrolysis in both species was found to be insensitive to ouabain (100 AM),
sodium azide (1 mM), sodium vanadate (100 AM) and h-glycerophosphate (10 mM) and was also found to depend on Ca2+ and Mg2+. V MAX
of the ectonucleotidase activity for guinea pig and mouse vas deferens was 958.4F66.3 and 79.7F8.5 pmol/min/mg, while K M was
625.1F45.2 and 406.0F29.0 AM, respectively. Cooling the tissues from 35 to 25 8C reduced the enzyme activity significantly ( Pb0.01) by
52.7F9.2% in guinea pig vas deferens and 34.9F5.3% in mouse vas deferens. ARL 67156 (100 AM), the specific ecto-ATPase inhibitor,
caused a reduction in enzyme activity in guinea pig and mouse vas of 54.1F16.4% and 53.0F7.6%, respectively ( Pb0.01). The degree of
inhibition of ATP hydrolysis by lowered temperature and 100 AM ARL 67156 correlates well with the reported potentiation and prolongation
of junction potentials under these conditions. It is concluded that ecto-ATPase or a closely related ectonucleotidase plays an important role in
the physiological regulation of purinergic neurotransmission.
Keywords: ATP; ARL 67156; Ecto-ATPase; Purinergic; Sympathetic; Temperature; Vas deferens
1. Introduction
The past 20–30 years has seen the role of purines,
particularly ATP, in neurotransmission being explored in
considerable detail. Certain aspects of purinergic transmission have been well studied and delineated. For
example, there is ample evidence to suggest that ATP
mediates the excitatory junction potentials, EJPs, and
excitatory junction currents, EJCs, occurring during neurotransmission at the sympathetic neuroeffector junctions of
rodent vas deferens (Sneddon and Burnstock, 1984;
Cunnane and Manchanda, 1989; Kennedy et al., 1996;
Sneddon, 2000; Ghildyal and Manchanda, 2002). It is
known that ATP acts via specific ligand-gated ion channels,
the P2X1–7 receptors (Khakh et al., 2001). However, other
aspects of purinergic neurotransmission are not equally well
elucidated, one of these being the inactivation of ATP
released as a neurotransmitter. Relatively little is known
about the mechanisms involved in the inactivation of this
neurotransmitter in the synapse and the effects of this
inactivation on the properties of the postjunctional
responses.
The presence and activity of a complex of synaptic
enzymes, namely synaptic ecto-ATPase (EC 3.6.1.3) and
nucleotidases is thought to curtail the actions of ATP and
limit its half-life in the synaptic cleft (Zimmermann et al.,
1998, 2000). The ectonucleotidases have been shown to be
present in a variety of tissues, and it has also been shown
that these enzymes can hydrolyse purine and pyrimidine diand triphosphates other than ATP (Ziganshin et al., 1995a;
Zimmermann, 1996; Caldwell et al., 1999). After its release
into the synapse, ATP is sequentially broken down to ADP
29
and AMP by the ecto-ATPase and ectonucleotidases and
finally to adenosine by the 5V-nucleotidase (Zimmermann et
al., 1998). Although it has been suggested that these
enzymes modulate purinergic transmission, the extent to
which they do so remains unclear. In particular, the ectoATPase, which is thought to be primarily responsible in
regulating the lifetime of ATP during transmission, is
incompletely explored.
Ecto-ATPase activity has been shown to be present in
many tissues and cell types ranging from blood cells to
neurons (Crack et al., 1995; Caldwell et al., 1999; Dunn et
al., 2001; Liang et al., 2000; Meghji and Burnstock, 1995;
Zinchuk et al., 1999a,b). At synapses, ecto-ATPase is
thought to hydrolyse the neuronally released ATP, degrading
it into ADP and inorganic phosphate (Pi). An early and
indirect line of evidence for this contention was the effect of
temperature on electrical activity during neurotransmission,
as seen at sympathetic neuromuscular junctions. Electrical
recordings from rodent vas deferens smooth muscle showed
that cooling the tissue significantly increased the amplitude,
rise time and time constant of decay of EJPs. The involvement of a temperature-dependent enzymatic process which
limited the activity of ATP at this synapse was invoked to
explain these observations (Cunnane and Manchanda, 1988;
Blakeley and Cunnane, 1982; Kuriyama, 1964). However,
there has been no direct biochemical evidence in this tissue
to show that ecto-ATPase indeed mediates the degradation
of ATP during neurotransmission, in a temperature-sensitive
manner.
More recently, the ATP analogue ARL 67156 has been
used as a specific inhibitor of the ecto-ATPase in a variety of
tissues and has been shown to prolong and potentiate EJPs
in the vas deferens (Sneddon et al., 2000). However, as in
the case of temperature, there is no direct evidence for the
inhibition of ecto-ATPase in the mammalian vas deferens by
ARL 67156. In this study, we have explored these questions
by conducting direct biochemical measurements of (a) the
effect of temperature on ATP degradation by the ectoATPase in the tissue and (b) the effect of the putative
inhibitor of synaptic ecto-ATPase, ARL 67156, on ATP
degradation. We discuss the significance of these results in
relation to the hypothesis that an ecto-ATPase sensitive to
inhibition by temperature and by ARL 67156 affects the
lifetime of ATP during purinergic neurotransmission at the
sympathetic neuromuscular junction.
2. Materials and methods
2.1. Chemicals
ATP used for the experiments was purchased from
HiMedia Laboratories, Mumbai, India. ARL 67156 was
obtained from Tocris, Bristol, UK. All other chemicals used
were of analytical grade purchased from SRL, Mumbai,
India. Both chemicals were dissolved in deionised water to
obtain 100-mM stock solutions. The stock solutions were
stored frozen till the time of use.
2.2. Animals
Male Dunkin–Hartley guinea pigs (300–700 g) or Swiss
mice (30–40 g) were used for the studies. The animals were
lightly anesthetized with sodium thiopentone (40 mg/kg1)
and killed by decapitation. The vasa deferentia were
dissected free and excess connective tissue carefully
removed. Pieces from the prostatic end of the vas deferens
were used for the enzyme assay.
2.3. Phosphate-release assay
The ecto-ATPase activity was assayed by using the
malachite green method for the determination of inorganic
phosphate, Pi (Chan et al., 1986). The assay was carried
out at 35 or 25F0.5 8C in 4-(2-hydroxyethyl)-1-piperazine
ethanesulphonic acid (HEPES) buffer solution containing
10 mM HEPES, 135 mM NaCl, 5.0 mM KCl, 2.0 mM
CaCl2, 2.0 mM MgCl2, 10 mM glucose adjusted to pH 7.4
and bubbled continuously with air. Temperature of the
assay medium was maintained in a temperature-controlled
shaken water bath. Small pieces, 1–2 mm in length
(weighing 2–3 mg for mouse, 5–10 mg for guinea pig),
from the prostatic end of the vas deferens were washed
and equilibrated in 0.5 ml HEPES buffer for 20–30 min.
The tissue pieces were then incubated with 0.5 ml of 100
AM ATP solution (for mouse) or 1 mM ATP solution (for
guinea pig) at 35 8C and the amount of phosphate liberated
was estimated at the end of 15 min. These concentrations
of ATP were found to be suitable in the respective tissues
based on existing data (Vizi et al., 2000) and pilot studies
performed to characterize the enzymatic activity, including
determination of the kinetic constant, K M. A second wash
was followed by incubation of the tissues with 0.5 ml
HEPES buffer at 25 8C or containing 100 AM ARL 67156
for 30 min. This was followed with incubation in 0.5 ml
buffer containing ATP (100 AM or 1 mM) at 25 8C (for
temperature studies) or along with 100 AM ARL 67156
(for ARL 67156 studies). Similarly, for studying the effect
of divalent cations on the enzyme activity, the tissues were
washed and incubated in Ca2+/Mg2+-free HEPES buffer to
which 1 mM EDTA was added. Samples were also tested
for enzyme activity in presence of ouabain (100 AM) and
sodium azide (1 mM; ecto-ATP diphosphohydrolase/ectoATPDase inhibitor), sodium vanadate (100 AM; inhibitor
of endo-ATPases) and h-glycerophosphate (10 mM;
inhibitor of nonspecific alkaline phosphatases) in order to
ascertain that the ATPase activity recorded was that of the
ecto-ATPase (Caldwell et al., 2001; Ziganshin et al.,
1995a). Because the experimental protocol involved a
double incubation, appropriate time controls were kept
throughout the duration of both incubations. These values
were subtracted from the test values to get the total
30
phosphate liberated due to the ATPase activity. The
amount of phosphate liberated due to the action of the
tissue pieces was calculated by interpolation from the
standard graph obtained by estimating phosphate in known
concentrations of KH2PO4 solutions. The amount of
phosphate liberated in the second incubation (in presence
of ARL 67156 or at 25 8C) was expressed as a percentage
of the first incubation (i.e., without ARL 67156 at 35 8C).
The kinetic constants (V MAX and K M) for the enzyme were
determined from the Lineweaver–Burk plot. The number
of experiments carried out has been indicated as n. Each
experiment was carried out in batches of 2–3 tissue
samples each. MeanFS.E.M. values were obtained from
pooled data from all batches. The Student’s paired t-test
was performed to determine the level of significance
between groups of samples.
3. Results
3.1. General properties of ectonucleotidase activity in
mouse and guinea pig vas deferens
ATP hydrolysis (100 AM and 1 mM) was found to be
linear for at least 60 min in both mouse and guinea pig vas
deferens tissues. The mean values of V MAX and K M as
determined from three sets of experiments were 79.7F8.5
pmol/min/mg and 406.0F29.0 AM, respectively in the
mouse vas deferens, and 958.4F66.3 pmol/min/mg and
625.1F45.2 AM, respectively in the guinea pig vas deferens.
In order to ascertain that the activity being measured was
that of the ecto-ATPase, we also measured the ATP
Fig. 1. A comparison of the relative velocities of inorganic phosphate (Pi)
production as a measure of the ectonucleotidase activity in guinea pig and
mouse vas deferens in presence of normal buffer solution containing Ca2+/
Mg2+ ions (control) and in Ca2+/Mg2+-free buffer+1 mM EDTA. Results
are shown as meanFS.E.M.; n=8 for guinea pig, n=6 for mouse ( Pb0.01;
paired t-test). The control activity indicates the total ectonucleotidase
activity in the tissue whereas the activity in absence of the divalent ions
represents the Ca2+/Mg2+-dependent ecto-nucleotidase activity in the tissue.
Table 1
Effect of endo-ATPase and phosphatase inhibitors on ATP hydrolysis
Compound/condition
Enzyme activity (percent of control, %)
Mouse
Guinea pig
Absence of ATP
Buffer preincubated with tissue
Sodium orthovanadate (100 AM)
Ouabain (100 AM)
Sodium azide (1 mM)
h – Glycerophosphate (10 mM)
Ca2+/Mg2+-free+EDTA (1 mM)
0
1.1F0.6
97.7F5.9
95.8F6.9
98.2F3.2
101.4F2.8
35.1F4.26
0
0.6F0.5
99.8F6.1
105.3F3.7
102F5.6
104.6F9.1
72.1F10.2
hydrolysis in presence of chemicals known to be inhibitors
of other types of ATPases. Ouabain (100 AM; Na+–K+
ATPase inhibitor), sodium azide (1 mM) and sodium
vanadate (100 AM; an ATP–ADP diphosphohydrolase
inhibitor) and h-glycerophosphate (10 mM; inhibitor of
nonspecific alkaline phosphatases) had no significant effect
on the measured ATPase activity (b3%), thus indicating that
the activity that is being reported is likely to be that of the
ecto-ATPase. The ecto-ATPase activity in the mouse and
guinea pig vas deferens was also characterized in terms of
its dependency on the divalent cations Ca2+ and Mg2+, as
Ca2+/Mg2+ dependence is one of the characteristic features
of the ecto-ATPase (Zimmermann et al., 1998; Ziganshin et
al., 1995a,b). The drop in ecto-ATPase activity in the
absence of Ca2+ and Mg2+ was found to be 61.9F10.2%
(n=8; Pb0.01) and 35.9F4.3% (n=6; Pb0.01) for the guinea
pig and mouse vas deferens, respectively (Fig. 1; Table 1).
The amount of Pi liberated due to spontaneous ATP
hydrolysis during the 15-min incubation periods was found
to be negligible (b2% of total Pi liberated). Similarly Pi
liberated by the tissue pieces during the period of assay was
also negligible (b0.5% of the total Pi liberated). As stated
above, these values were subtracted from the total Pi
liberated in order to obtain accurate estimates of the
ectonucleotidase activity.
Fig. 2. Effect of cooling on relative velocity of inorganic phosphate (Pi)
production in guinea pig and mouse vas deferens. Relative ectonucleotidase
activities in control (at 35 8C) and after cooling (at 25 8C) are shown as
meanFS.E.M.; n=8 for guinea pig, n=14 for mouse ( Pb0.01; paired t-test).
31
Fig. 3. Comparison of the effects of ecto-ATPase inhibitor, ARL 67156, on
inorganic phosphate (Pi) production in guinea pig and mouse vas deferens
is shown. Relative ecto-ATPase activities in absence (control) of and
following 30 min incubation with ARL 67156 (100 AM) are shown as
meanFS.E.M.; n=13 for guinea pig, n=8 for mouse ( Pb0.01; paired t-test).
3.2. Effect of temperature on ectonucleotidase activity in
guinea pig and mouse vas deferens
The total ectonucleotidase activity measured in wet tissue
pieces of mouse and guinea pig vas deferens was found to
decrease significantly with lowered temperature. With a 10
8C drop in temperature, from 35 to 25 8C, a 52.7F9.2%
drop in ecto-ATPase activity was observed in the guinea pig
vas deferens (n=8; Pb0.01). Similarly for the mouse vas
deferens the drop in enzyme activity was 34.9F5.3% (n=14;
Pb0.01). See Fig. 2.
The Q 10 for the change in enzyme activity was calculated
as (Enzyme activity at X 8C)/(Enzyme activity at (X10
8C)). The Q 10 values for the guinea pig and mouse vas
deferens enzyme activity were 2.1 and 1.5, respectively.
3.3. Effect of ARL 67156 on ecto-ATPase activity in guinea
pig and mouse vas deferens
ARL 67156 (100 AM), which has been reported to be a
specific ecto-ATPase inhibitor (Sneddon et al., 2000; Westfall et al., 2000a), was found to cause a reduction in enzyme
activity in guinea pig and mouse tissues by 54.1F16.4%
(n=13) and 53.0F7.6% (n=8), respectively ( Pb0.01; Fig.
3). These results therefore indicate that this fraction of the
ectonucleotidase activity is contributed by the ecto-ATPase
in the tissue.
4. Discussion
Although a physiological role for the ectonucleotidases
in general and the ecto-ATPase in particular has been
suggested in the modulation of purinergic neurotransmis-
sion, certain aspects of this role remain unclear. The effects
of cooling and of the specific ecto-ATPase inhibitor, ARL
67156, on EJPs have been suggested to be consistent with a
role of the ecto-ATPase in modulating purinergic neurotransmission in the rodent vas deferens (Cunnane and
Manchanda, 1988; Westfall et al., 1996). However, there
have been no reports of direct estimations of the activity of
this enzyme in the above conditions, i.e., change of
temperature and exposure to ARL 67156, in the rodent
vas deferens. Our results and their relevance to our
understanding of synaptic inactivation of ATP and its effect
on purine-mediated synaptic potentials are discussed below.
We have measured the inorganic phosphate liberated
during the breakdown of ATP. The Pi measured in our assay
could be due to the sequential break down of ATP to ADP,
AMP and adenosine by the ecto-ATPase and a host of
ectonucleotidases and extracellular phosphatases present in
the vas. Our results using enzyme inhibitors that target
ATPases other than the ecto-ATPase (e.g., ecto-ATP
diphosphohydrolase and endo-ATPases) indicate that under
the conditions of our experiments, the principal pathway for
ATP hydrolysis being monitored is that of the ectonucleotidase, namely, ecto-ATPase, ecto-ADPase and 5V-nucleotidase. The contribution of ecto-ATPase versus the other
nucleotidases to Pi liberation in our experiments dealing
with the effects of temperature is unknown. However, in our
experiments with ARL 67156, because ARL 67156 is a
specific inhibitor of ecto-ATPase, the inhibition of ATP
hydrolysis observed could be attributed to the ecto-ATPase
alone. This is further supported by the fact that the extent of
inhibition observed in our studies (based on Pi detection) is
similar to that observed in studies with ARL 67156 where a
more direct method of ATP detection was used (Kennedy et
al., 1997). In the following discussion, therefore, while we
refer to ectonucleotidase activity in general for the
characterization and temperature-sensitivity of the enzyme,
we refer more particularly to ecto-ATPase activity when
discussing results with ARL 67156.
4.1. Characterization of ectonucleotidase
The enzyme activity measured was found to be insensitive to inhibitors of other well known ATPases. ATP
hydrolysis was found to be unaltered in presence of ouabain,
sodium azide and sodium vanadate and h-glycerophosphate,
thus ruling out the involvement of intracellular ATPases and
non-specific phosphatases in the hydrolysis of ATP (Ziganshin et al., 1995a; Caldwell et al., 2001). We have therefore
inferred that the enzyme activity measured in the present
experiments on rodent vas deferens was likely to be that of
the synaptic ecto-ATPase.
We have shown the dependency of the enzyme activity
on divalent cations like Ca2+ and Mg2+. In the guinea pig
vas deferens, a 72.44% drop in enzyme activity was
observed on removal of Ca2+/Mg2+ from the medium,
whereas in the mouse, the activity drop was 34.8%. Thus,
32
the inhibition in the absence of Ca2+/Mg2+ is incomplete.
Although one of the characteristics of the ecto-ATPase is its
dependence on divalent cations like Ca2+ and Mg2+, various
studies have also revealed Ca2+/Mg2+-independent forms of
this enzyme, for example, in Xenopus oocytes, endothelial
cells and synaptic plasma membranes (Ziganshin et al.,
1995a; Meghji and Burnstock, 1995). This suggests the
presence of isoforms of the enzyme with varying dependence on Ca2+/Mg2+.
4.2. Temperature-sensitivity and its significance
Earlier electrophysiological studies on the vas deferens
have shown alterations in the properties of synaptic
potentials, i.e., the intracellularly recorded EJPs and the
extracellularly recorded EJCs, upon cooling the tissue
(Kuriyama, 1964; Blakeley and Cunnane, 1982; Cunnane
and Manchanda, 1988). These alterations (e.g., increase of
amplitude, rise time and time constant of decay) of electrical
events were thought to be consistent with the idea of
inhibition of a putative temperature-sensitive synaptic
inactivation mechanism for the neurotransmitter, most
likely, ATP. Furthermore, the EJP-like ATP potentials were
affected very similarly by temperature. In contrast, the
potentials generated by similar application of a,h-methylene
ATP (a nondegradable analogue of ATP) were found to be
unaltered by cooling (Cunnane and Manchanda, 1988).
Because a,h-methylene ATP is resistant to degradation by
ecto-ATPase, changes in the level of ATPase activity, for
example, brought about by changing temperature, would not
influence the responses to a,h-methylene ATP. These
findings were consistent with the modulation of the
amplitude and time course of EJPs and with ATP-potentials
by a temperature-sensitive enzyme in the tissue. The
presence and the actions of such an enzyme at that time
had not been demonstrated and the authors had hinted at the
possibility of the presence of a synaptic enzyme, inhibition
of which upon cooling could result in the changes observed
in the EJPs, EJCs and ATP potentials.
An ecto-ATPase activity similar to the ectonucleotidase
activity characterized here has been demonstrated in a
variety of tissues like the chicken gizzard, Xenopus oocyte,
skeletal muscle t-tubules, etc. (Ziganshin et al., 1995b;
Caldwell et al., 1999, 2001; Megias et al., 2001). This ectoATPase activity was shown to be temperature sensitive.
However, in the rodent vas deferens, this temperature
sensitivity has not yet been shown and it is not known
whether this property is in qualitative and quantitative
agreement with the observed effects of temperature on
synaptic potentials. The present results show that there is
indeed a temperature-sensitive ecto-nucleotidase activity (a
large part of which is likely to be contributed by the ectoATPase—see Results on characterization) present in the
rodent vas deferens. Furthermore, the extent of inhibition
following a 10 8C reduction in temperature was found to be
in the range of 52% and 35% in the guinea pig and mouse
vas deferens, respectively, which is consistent with the
extent of change seen for the electrical events. In Table 2,
we compare the Q 10 values of the observed ectonucleotidase
activity and those reported for the rise times and decays of
the various electrical events in the guinea pig vas deferens.
It is evident from Table 2 that the Q 10 for ectonucleotidase activity in the guinea pig vas deferens falls in the
range of those reported earlier for the electrical activities in
this tissue, being especially close to the Q 10 for EJCs. The
close correspondence between the Q 10 of the ectonucleotidase and that of the EJC suggests that these enzymes indeed
play a major role in determining neurotransmitter lifetime in
the synaptic cleft during neurotransmission. It should be
noted that EJCs are more accurately thought to reflect the
time course of neurotransmitter action because they are
generated directly by transmitter-activated membrane currents without modulation by passive electrical properties as
in the case of the EJPs (Cunnane and Manchanda, 1989).
The time course of ATP potentials involve many more
factors in addition to those involved in generation of the
EJPs, e.g., distance of the application microelectrode from
the tissue surface, concentration of ATP applied, etc. Our
results, therefore, demonstrate the temperature sensitivity of
the ectonucleotidase responsible for inactivating neuronally
released ATP. They lend support to the earlier hypothesis of
a temperature-dependent enzymatic inactivation mechanism
which was thought to underlie the effects of temperatureinduced alterations of junctional potentials and currents
(Cunnane and Manchanda, 1988). The Q 10 value for the
measured ectonucleotidase activity in mouse vas deferens
was found to be 1.5. This value was quite similar to that
reported for the Q 10 value of ecto-ATPase activity in the
Xenopus oocytes (Ziganshin et al., 1995b).
4.3. Inhibition of ecto-ATPase by ARL 67156
ARL 67156, an analogue of ATP, has been reported to
have a higher specificity for the ecto-ATPase over the
purino-receptors, than other known compounds (Kennedy et
al., 1996). It has been reported to inhibit the ecto-ATPase in
a concentration-dependent manner in blood cells, rat tail
Table 2
Q 10 for ectonucleotidase activity and electrical events in guinea pig vas
deferens
Electrical events
EJPs
EJCs
ATP potentials
Ectonucleotidase activity
Q 10 valuesa
Rise time
decay time
1.6
2.1
2.9
1.8
2.27
2.95
2.1
(Cunnane and Manchanda, 1988; present results).
a
Q10 for ectonucleotidase activity ¼
Q10 for electrical events ¼
ATP degradation at 258C
ATP degradation at 358C
rise=decay time at 358C
:
rise=decay time at 258C
33
artery, vas deferens, chromaffin cells, etc. (Crack et al.,
1995; McLaren et al., 1998; Westfall et al., 2000a,b;
Drakulich et al., 2004). Most studies in smooth muscle
organs, suggesting the inhibition of ecto-ATPase by ARL
67156, have been carried out on the physiological responses
of these organs. Enhanced purinergic neurotransmission,
due to inhibition of ecto-ATPase by ARL 67156, was
suggested based on studies showing increased amplitudes of
EJPs (Sneddon et al., 2000) and enhanced ATP-evoked
contractions in presence of ARL 67156, in the guinea pig
vas deferens and rabbit ear artery (Westfall et al., 1996,
1997). These studies had therefore suggested that the actions
of ATP as a sympathetic neurotransmitter were modulated
by the presence and actions of the ecto-ATPase. Our results
with the effects of ARL 67156 on guinea pig vas deferens
help to explain the changes in physiological responses
described above.
A recently discovered factor that could conceivably
contribute to our findings is the releasable form of the
ecto-ATPase, which is released in a stimulation-dependent
manner from pre-synaptic nerve terminals (Kennedy et al.,
1997). This releasable ecto-ATPase has also been shown to
be inhibited by ARL 67156 (Westfall et al., 2000a,b;
Mihaylova-Todorova et al., 2002). Its release is found to be
greatest at higher frequencies of stimulation, i.e., around 8
Hz; whereas at 2 Hz, it release is significantly lower
(Westfall et al., 2000a). At the frequencies used to elicit the
EJPs (0.6–1 Hz), the release pattern and therefore the
contribution of this form of the ecto-ATPase in the
modulation of purinergic neurotransmission has not yet
been demonstrated. It should be noted, however, that our
study does not involve stimulation of the vas deferens.
Moreover, the buffer solution in which the tissue was
incubated for 30 min did not show an ATPase activity (see
Table 1). Hence, it seems unlikely that the releasable ectoATPase might interfere with or add to the measured enzyme
activity, which we suppose is of the membrane-localized
form of the ecto-ATPase.
5. Conclusion
We have demonstrated the presence and activity of an
ectonucleotidase in guinea pig and mouse vas deferens,
closely resembling the becto-ATPaseQ activity which has
been characterized in a number of other reports. We have
also shown that both physical and pharmacological interventions, in the form of changes of temperature and
exposure to ARL 67156, respectively, directly affect ATP
hydrolysis in the rodent vas deferens by inhibiting the
enzyme(s) involved. Furthermore, these results correlate
well with previous physiological studies and lend support to
the suggestion that ecto-ATPase inhibition underlies the
changes observed in smooth muscle physiological
responses, such as junction potentials and contractions
under similar conditions. Our study thus strengthens the
contention that ATP acts as a neurotransmitter in the vas
deferens and that the synaptic ecto-ATPase modulates the
presence and activity of ATP during neurotransmission at
this neuromuscular junction. The ecto-ATPase might similarly regulate purinergic neurotransmission at other synapses by shaping the ATP-generated synaptic potentials and
contractions, where its actions deserve to be elucidated in
greater detail.
Acknowledgements
We acknowledge the financial support provided by the
Department of Science and Technology (DST), India (SP/
SO/B-11/2000), and by IIT-Bombay under the Cross
Disciplinary Research Grant. P.G. was supported by grants
from the Indian Council for Medical Research (BMS-45/
2000) and the Lady Tata Memorial Trust.
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