0022-3565/02/3032-439 –444$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 303:439–444, 2002
Vol. 303, No. 2
35113/1021259
Printed in U.S.A.
Perspectives in Pharmacology
ATP as a Cotransmitter in Sympathetic Nerves and Its
Inactivation by Releasable Enzymes
DAVID P. WESTFALL, LATCHEZAR D. TODOROV, and SVETLANA T. MIHAYLOVA-TODOROVA
Department of Pharmacology, University of Nevada School of Medicine, Reno Nevada
Received May 31, 2002; accepted August 12, 2002
The evidence is now substantial that ATP plays a role in
sympathetic neuroeffector mechanisms as a cotransmitter
with norepinephrine (NE) (Stjarne, 1989; Westfall et al.,
1991; Silinsky et al., 1998; Burnstock, 1999). Much of the
early evidence implicating ATP as a cotransmitter came from
studies of the rodent vas deferens, and work in the authors’
laboratory, as well as by others, has contributed to this
knowledge (see Sneddon and Westfall, 1984). This article will
briefly review the current understanding of ATP and NE
cotransmission using the vas deferens as a model and, further, will discuss more recent information about an unusual
mechanism that links inactivation of ATP to nerve stimulation-induced release of degrading enzymes.
Research referenced from the authors’ laboratory was supported by National Institutes of Health Grants HL 38126 and NS 08300 and a grant from
the Foundation for Research.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.035113.
to ADP, AMP, and adenosine, thereby terminating the action of
ATP. In the guinea pig vas deferens, there appear to be at least
two enzymes, one that converts ATP to ADP and ADP to AMP
(an ATPDase) and a second enzyme that converts AMP to
adenosine (an AMPase). An important feature of this process is
that the transmitter-metabolizing nucleotidases are released
into the synaptic space as opposed to being fixed to cell
membranes. A preliminary characterization of these enzymes
suggests that the releasable ATPDase exhibits some similarities to known ectonucleoside triphosphate/diphosphohydrolases, whereas the releasable AMPase exhibits some similarities to ecto-5⬘-nucleotidases.
Cotransmission in Vas Deferens
The vas deferens is supplied with a dense sympathetic
innervation, and stimulation of the nerves results in a biphasic mechanical response that consists of an initial rapid
twitch, followed by a maintained contraction (see Westfall
and Westfall, 2001). The evidence is now clear that the first
phase of the response is mediated mainly by ATP acting on
postjunctional P2X receptors, whereas the second phase is
mediated mainly by NE acting on ␣1-adrenoceptors (Fig. 1).
Activation of P2X receptors by ATP results in the production
of excitatory junction potentials that summate to produce
action potentials, which then propagate from one smooth
muscle cell to another (Sneddon et al., 1982; Sneddon and
Westfall, 1984). The calcium influx that results from depolarization leads to the generation of a major component of the
first phase of the contraction. The activation of the ␣1-adrenoceptors by NE results mainly in the second maintained
phase of the contraction, by a mechanism that involves the
release of intracellular calcium by a phosphoinositide path-
ABBREVIATIONS: NE, norepinephrine; NPY, neuropeptide Y; ENPPases, ectonucleotide pyrophosphatase/phosphodiesterases; ENTPDases,
ectonucleoside triphosphate/diphosphohydrolases; EFS, electrical field stimulation; ADO, adenosine; ARL 67156, 6-N-N-diethyl-␤,␥-dibromomethylene-D-ATP.
439
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ABSTRACT
ATP and norepinephrine (NE) are cotransmitters released from
many postganglionic sympathetic nerves. In this article, we
review the evidence for ATP and NE cotransmission in the
rodent vas deferens with special attention to the mechanisms
involved in removing the cotransmitters from the neuroeffector
junction. Although the clearance of NE is well understood (e.g.,
the primary mechanism being reuptake into the nerves), the
clearance of ATP is just beginning to be explained. The general
belief has been that ATP is metabolized by cell-fixed ectonucleotidases. It now seems, however, that when ATP is released from nerves as a transmitter there is a concomitant
release of nucleotidases that rapidly degrade ATP sequentially
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Westfall et al.
way (Khoyi et al., 1988). The cotransmitters are apparently
released from the same types of nerves because pretreatment
with 6-hydroxydopamine, an agent that specifically destroys
adrenergic nerves, abolishes both phases of the neurogenically induced biphasic contraction (Fedan et al., 1981).
In addition to ATP and NE, neuropeptide Y (NPY) has been
found in some sympathetic nerves, and the release of NPY,
along with ATP and NE, has been demonstrated in response
to nerve stimulation of the guinea pig vas deferens (Kasakov
et al., 1988). Although it is clear that ATP and NE are
cotransmitters in the vas deferens, being responsible for the
phasic and tonic contractions of the smooth muscle, NPY
seems to play a modulatory role by influencing the release
and the postjunctional actions of ATP and NE (Stjarne et al.,
1986; Bitran et al., 1991).
Prejunctional Modulation of Cotransmitter
Release
Probably all nerves that release NE have prejunctional
␣2-adrenoceptors that, when stimulated, reduce the release
of NE. Stimulation of these “autoreceptors” in the vas deferens of the mouse, rat, and guinea pig reduce not only the
release of NE but also the release of ATP (Brown et al., 1983;
Sneddon and Westfall, 1984; Driessen et al., 1993). Endogenously released NE, however, has a greater influence on its
own release than that of ATP. Interestingly, certain ␣2-adrenoceptor agonists (e.g., xylazine) produce a greater reduction
of ATP release than of NE release (Westfall et al., 1996a).
These results suggest that the release of ATP and NE can be
differentially regulated, and therefore, the cotransmitters
may be differentially released (vide infra).
ATP also seems to function as a neuromodulator, causing a reduction of transmitter release in the peripheral and
central nervous systems (Silinsky et al., 1998; Stone et al.,
2000). The ability of antagonists of P1 receptors (receptors
for adenosine) to reduce the inhibition of transmitter release by ATP has supported the popular view that the
effect of ATP is caused by its metabolism to adenosine,
which then activates P1 receptors (Kirkpatrick and Burnstock, 1992). Adenosine has long been known to reduce
transmitter release in vas deferens (Hedqvist and Fredholm, 1976) and other tissues (see Paton, 1981). Although
it is likely that a portion of the effect of ATP is due to the
formation of adenosine, there is intriguing evidence indicating that nucleotides can act per se to inhibit transmitter
release without first being degraded to adenosine (Shinozuka et al., 1988; von Kugelgen et al., 1989; Forsyth et al.,
1991; Todorov et al., 1994b). The specifics of the receptor
type upon which ATP acts directly have not been fully
elucidated. Suggestions range from a P2Y type (von Kugelgen et al., 1989; von Kugelgen and Starke, 1991) to a
unique hybrid receptor with some features of both P1 and
P2 receptors. Shinozuka et al. (1988) have referred to this
receptor as a P3 receptor. This concept of prejunctional
autoreceptors is shown schematically in Fig. 1.
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Fig. 1. Schematic representation of NE and ATP cotransmission in the guinea pig vas deferens. NE and ATP are stored in and released from
postganglionic sympathetic nerves, most likely from separate neurotransmitter vesicles. NE acts on postjunctional ␣1-adrenoceptors on smooth muscle
cells that mediate, via the release of Ca2⫹ from intracellular stores and also possibly by Ca2⫹ influx, the tonic component (b) of the neurogenically
induced [electrical field stimulation (EFS)] biphasic contraction. NE can also act on prejunctional ␣2-adrenoceptors that modulate the release of
neurotransmitter. The major mechanism for clearing NE from the neuroeffector junction involves reuptake via the action of a specific neuronal
transporter (T). ATP acts on postjunctional P2X receptors which promote Ca2⫹ influx and is responsible for the phasic component (a) of the biphasic
contraction. ATP is cleared by being sequentially metabolized to ADP, AMP, and adenosine (ADO) via the action of releasable nucleotidases. The
enzymes seem to be released in parallel with ATP and from a similar site. At least two separate enzymes seem to be involved in the metabolism of
ATP; a releasable nucleoside triphosphate/diphosphohydrolases-like enzyme (r NTPDase) that breaks down ATP to ADP and AMP and a releasable
5⬘-nucleotidase (r 5⬘N) that breaks down AMP to ADO. Cell attached, ecto-triphosphate/diphosphohydrolase (E-NTPDase) and ecto-5⬘-nucleotidase
(E-5⬘N) may also contribute to the metabolism of ATP, but their contribution is slight in comparison to the releasable nucleotidases. Adenosine can
modulate neurotransmitter release by acting on prejunctional P1-receptors. ATP may also modulate neurotransmitter release directly by acting on
P2Yor P3 receptors (not shown; see text for further discussion).
Metabolism of ATP by Releasable Nucleotidases
Release of the Cotransmitters
Inactivation of Transmitters
For neurotransmission to be effective, the neurotransmitters, once released, need to be inactivated or removed from
the neuroeffector junction. For amine transmitters, such as
NE, dopamine, and serotonin, the mechanism by which this
occurs is well understood. The transmitters are taken back
up into the nerve by specific high-affinity transporters that
clear the amines from the synapse (see review by Amara and
Kuhar, 1993). The amine transporters are important drug
targets in that a number of clinically useful antidepressants
and antianxiety drugs have as their mechanism of action
inhibition of neuronal reuptake.
A different process terminates the action of the neurotransmitter acetylcholine. The enzyme acetylcholinesterase,
which is associated with pre- and postjunctional membranes,
forms a complex with acetylcholine and reacts to release
choline, which is then taken up by the nerve. Drugs that
inhibit the activity of acetylcholinesterase have important
therapeutic uses in myasthenia gravis, glaucoma, paralytic
ileus, atony of the urinary bladder, and enhancing cognition
in victims of Alzheimer’s’ disease (Taylor, 2001).
The mechanism of clearance of ATP from the synapse is not
as well understood as the mechanisms for removal of the
autonomic neurotransmitters NE and acetylcholine. The general belief has been that ATP, once it is released into the
neuroeffector junction, is metabolized by extracellularly directed, membrane-bound nucleotidases to ADP, AMP, and
adenosine (Gordon, 1986; Zimmermann, 1992; Plesner,
1995). The rate of metabolism of ATP by these ecto-ATPases
is relatively slow compared with synaptic events (Pearson et
al., 1985; Plesner, 1995). Work by Todorov et al. (1996) suggested that a process other than metabolism of ATP by membrane-bound ecto-ATPases may be involved in terminating
the action of ATP. For example, superfusion of the sympathetically innervated vas deferens preparation with ATP revealed that, over a 1 minute superfusion period, there was
essentially no metabolism of the nucleotide, although metabolism of ATP would have been expected if ecto-ATPases were
the primary inactivation mechanism. If the sympathetic
nerves were stimulated during the superfusion period, however, there was virtually complete degradation of ATP
(Todorov et al., 1997). This phenomenon is illustrated in Fig.
2.
Releasable Nucleotidases
The nerve stimulation-related metabolism of ATP is associated with the release of nucleotidases that breakdown ATP,
as well as ADP and AMP, to adenosine. The enzyme activity
that overflows the tissue preparation during nerve stimulation remains stable in the superfusate and has allowed a
preliminary characterization of the kinetics and sensitivity
to antagonists of these nucleotidases (Westfall et al., 2000a,b;
Mihaylova-Todorova et al., 2002).
Inhibition of the propagation of action potentials with tetrodotoxin, suppression of postganglionic sympathetic neurotransmission with guanethidine, or inhibition of exocytosis by omission of extracellular Ca2⫹ all prevented the release of
nucleotidase activity, strongly suggesting that the sympathetic
nerves are the source of the enzymes (Todorov et al., 1997).
Interestingly, the time course of release and modulation of
release by prejunctional receptors of the nucleotidases indicates
that the enzyme activity is coreleased with ATP and not with
NE (Mihaylova-Todorova et al., 2001). These results suggest
that the proteins carrying the enzyme activity originate from
ATP-storage vesicles as opposed to catecholamine vesicles.
At this point, it is not known whether these nucleotidases
represent a heretofore unidentified type of enzyme or whether
these are known enzymes and the ability to be released and to
metabolize transmitter ATP are newly recognized features. In
an attempt to clarify this issue, a number of known nucleotidases have been considered as candidates. For example, there
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Even though there is now a consensus that neurotransmission commonly involves the release of several neurotransmitter substances (see Furness et al., 1989; Hokfelt et al., 1992;
Lundberg, 1996), questions remain about whether storage
and release of cotransmitters occur in or from common sites.
In the case of the sympathetic cotransmitters ATP and NE,
the initial concept was based on an analogy with adrenal
chromaffin cells that release ATP and catecholamines in the
same ratio in which they are stored in the chromaffin secretory granules. Consequently, it has been assumed that the
sympathetic nerves also store ATP and NE in the same
synaptic vesicles, and therefore, upon release, the two cotransmitters are presented at their specific receptors simultaneously and in constant proportions (Stjarne, 1989, 1994;
Brock et al., 1997; Brock and Cunnane, 1999).
When the time courses of release of endogenous ATP and
NE from the sympathetic nerves of vas deferens and the time
courses of release of ATP and catecholamines from adrenal
chromaffin cells were compared, different patterns were observed (Todorov et al., 1994a, 1996). Adrenal chromaffin cells
release ATP and catecholamines continuously and in a constant molar ratio. The sympathetic nerves, on the other
hand, release ATP transiently only at the beginning of a train
of nerve stimulation. The release of NE occurs later in the
train and, once started, is maintained throughout the course
of nerve stimulation (Todorov et al., 1996; MihaylovaTodorova et al., 2001). These findings with sympathetic
nerves were, to our knowledge, the first direct evidence that
ATP and NE are not always released simultaneously. It
seems that the cotransmitter composition of the “cocktail” of
neurotransmitters released from the sympathetic nerves
changes during the course of stimulation. Early in the train,
the cocktail contains mainly ATP, ATP, and NE in the middle
and almost exclusively NE near the end of the train of stimuli. In addition, there is a good correlation between this
dissociated temporal pattern of release of cotransmitters and
the temporal pattern of the mechanical response of the tissue. The purinergic twitch contraction of the vas deferens
seems to reflect the early and transient release of ATP,
whereas the adrenergic tonic contraction reflects the delayed
and sustained release of NE. To explain the temporal disparity of the release of the cotransmitters, we have suggested
that the sympathetic nerves may store ATP and NE in separate vesicles and release them via independent mechanisms
(Todorov et al., 1994a, 1996). Recently, the concept of separate storage and differential release of the cotransmitters
ATP and NE has been receiving growing support (Brock et
al., 2000; Stjarne, 2001).
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Westfall et al.
are nucleotidases that might be expected to be involved in some
way in the process of neurotransmission, such as the vacuolar
H⫹-transporting ATPase, the Na⫹/K⫹-ATPase, the multidrug
resistance channel, and the cytosolic N-ethylmaleimide-sensitive fusion protein. All of these, however, have been rejected as
candidates based on the fact that specific antagonists, namely
bafilomycin, ouabain, orthovanadate, and N-ethylmaleimide
failed to affect the activity of the releasable nucleotidases
(Todorov et al., 1997; Fig. 3).
There are a variety of other enzymes that have the potential to dephosphorylate extracellular nucleotides, including
phosphatases, nucleotide pyrophosphatases, phosphodiesterases, and ecto-nucleotide triphosphate diphosphohydrolases.
Most of these enzymes are fixed to cell membranes with the
catalytic site facing the extracellular space where they can
metabolize extracellular nucleotides and are, therefore, referred to as ecto-enzymes. There is also evidence that some
ectophosphatases could be released from cell membranes
upon activation of endogenous phospholipases and cleavage
of a glycosylphosphatidyl-inositol linkage anchoring the protein to the cell membrane (Hooper, 1997).
In an attempt to more completely understand the nature of
the releasable nucleotidases, Mihaylova-Todorova et al.
(2002) examined the effects of several pharmacological
agents known to inhibit various ecto-enzymes. Known inhibitors of phosphatases, such as levamisole and phosphatase
inhibitor cocktail II (Sigma-Aldrich, St. Louis, MO), however,
do not affect the activity of the releasable nucleotidases (Fig.
3). Also, 3-isobutyl-1-methylxanthine, a nonspecific phosphodiesterase antagonist, failed to inhibit the ATPase and
AMPase activities of the releasable nucleotidases. Additionally, para-nitrophenyl thymidine monophosphate, a preferred substrate of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPases) had no influence on the ATP
metabolism by releasable nucleotidases indicating that ENPPases do not contribute to this phenomenon (Fig. 3).
The releasable nucleotidases seem to have some similarities with members of the mammalian ecto-ATPase CD39
gene family. It has recently been suggested that this family of
enzymes be referred to as ENTPDases (Zimmermann et al.,
Fig. 3. Pharmacological characterization of releasable nucleotidases. The
effects of various known nucleotidase inhibitors were tested on the soluble ATPase (solid bars) and AMPase (hatched bars) activities that overflow the guinea pig vas deferens tissue preparations during nerve stimulation using the fluorescent “etheno” analogs of ATP and AMP as initial
substrates. Both ATPase and AMPase activities are abolished when the
free Ca2⫹ concentration of the solution is decreased in the presence of
EGTA, or the pH of the solution is decreased below 4 by addition of
perchloric acid. Suramin, a known ATPase inhibitor, suppresses the
releasable ATPase activity but has no effect on the AMPase activity.
Another drug promoted as an ATPase inhibitor, ARL 67156, suppresses
both activities, whereas sodium aside has no effect. Known 5⬘-nucleotidase inhibitors [␣,␤-methylene ADP (␣,␤ m-ADP) or concanavalin A]
abolish the AMPase activity but have no effect on the ATPase activity.
Inhibitors of acid and neutral phosphatases, tyrosine protein phosphatases (protease inhibitor cocktail II; Sigma-Aldrich), alkaline phosphatase (levamisole), or ecto-phosphodiesterases (IBMX) have no affect on
either ATPase or AMPase activities. The ATPase or AMPase activities
were also not affected by the presence of para-nitrophenyl timidine 5⬘monophosphate (pnp-TMP), a specific substrate for ENPPases. The figure
is based on data presented by Todorov et al. (1997) and MihaylovaTodorova et al. (2002).
2000). An example of a similarity is that, 6-N-N-diethyl-␤,␥dibromomethylene-D-ATP (ARL 67156) inhibits the activity
of ecto-ATPases expressed by blood (Crack et al., 1995) and
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Fig. 2. Time course of metabolism of exogenous ATP by the isolated vas deferens of the guinea pig under resting experimental conditions (A) and
during neurogenic stimulation (B). Guinea pig vas deferens tissue preparations are superfused at a rate of 2 ml/min with Krebs’ solution containing
the fluorescent “etheno” analog of ATP (e-ATP) at a concentration of 0.1 ␮M. The amounts of e-ATP and its metabolites e-ADP, e-AMP and
etheno-adenosine (e-ADO) are determined in consequently collected samples of the superfusing solution by a high-performance liquid chromatography-based assay (Todorov et al., 1996). The abscissa in both A and B shows the 10-s intervals in which samples are collected: Krbs indicates a control
sample of the Krebs’ solution used for superfusion of the tissues preparations; PS indicates samples of the superfusate collected for 10 s immediately
before application of a neurogenic, EFS; S1 through S6 indicate samples collected during EFS, and AS indicates samples collected after the stimulation.
There is a little degradation of the exogenously supplied e-ATP by vas deferens under resting experimental conditions, as shown by the slight decrease
in the amount of e-ATP and the increase of its metabolites e-ADP, e-AMP, and e-ADO (compare Krebs and PS). The same low level of metabolism is
maintained in the absence of nerve stimulation (A). In contrast, during nerve stimulation there is a substantial, almost complete degradation of e-ATP
to e-ADO (B). The figure is based on data presented by Todorov et al., 1997.
Metabolism of ATP by Releasable Nucleotidases
Implications for Drug Development
Extracellular adenine nucleotides and nucleosides are now
known to be involved in a plethora of physiological functions
and pathological conditions including neurotransmission and
neuromodulation, platelet aggregation and hemostasis, pulmonary function, nociception, and auditory and ocular function to name a few (Abbracchio and Burnstock, 1998; Burnstock and Williams, 2000). As the diverse actions of purines
have become increasingly recognized, there has been a growing interest in how they produce their effects. This has lead
to an active investigation of the cell surface receptors upon
which the purines act as well as of potentially useful agonists
and antagonists of adenosine receptors and P2 receptors. In
addition to receptor agonists and antagonists, another phar-
macological approach, which has received less attention,
would be to develop agents that would affect the extracellular
concentration of the endogenous purines by influencing their
metabolism.
A good example of this latter approach relates to platelet
aggregation. As pointed out by Zimmermann (1999), there is
growing evidence for a specific ecto-ATPase of the CD39 gene
family associated with endothelial cells that limits the extent
of platelet aggregation by converting ADP, which induces
platelet aggregation, to adenosine, which has antiaggregation activity (Kaczmarek et al., 1996; Marcus et al., 1997).
The hypothesis that CD39/NTPDase 1 is a key thromboregulatory factor has been supported by in vivo experiments with
NTPDase 1-null (CD39 ⫺/⫺) mice (Enjyoji et al., 1999; Imai
et al., 1999). Furthermore, a recombinant form of this ectoATPase, which is soluble, has been shown to block ADPinduced platelet aggregation in vitro and has potential as a
therapeutic agent for patients with thromboembolic disorders (Gayle et al., 1998).
There may be situations where it is useful to enhance
purinergic neurotransmission, just as it is for adrenergic,
dopaminergic, serotonergic, and cholinergic neurotransmission. In this regard, Westfall et al. (1996b, 1997) have shown
that ARL 67156 enhances neurotransmission in vas deferens
and urinary bladder presumably, in part, by preventing the
rapid breakdown of ATP by the neuronally released nucleotidases. There is also evidence that sympathetic nerves in
blood vessels release nucleotidases (e.g., rat caudal and rabbit saphenous artery; L. D. Todorov and S. T. MihaylovaTodorova, unpublished results). Thus, neurotransmission in
blood vessels may be influenced by pharmacological manipulation of the nucleotidases. As more is learned about the
releasable nucleotidases and as specific inhibitors are developed, one might expect the emergence of a class of drugs that
will be to purinergic neurotransmission what amine reuptake inhibitors are to adrenergic and serotonergic neurotransmission and what cholinesterase inhibitors are to cholinergic neurotransmission. In addition to potential
therapeutic applications for enzyme inhibitors, there may be
a place for recombinant forms of the neuronal-releasable
nucleotidases, in analogy with the recombinant CD39 ectoATPase being investigated as an antiplatelet aggregatory
agent.
As a closing thought, it may be fortuitous that there are
multiple types of ecto- and releasable nucleotidases. This
provides some hope that system specific enzymes and inhibitors can be developed.
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