The autonomic nervous system regulates smooth muscle contractility through both sympathetic
and parasympathetic influences. In some tissues, such as the urinary bladder, parasympathetic
influences predominate and nerves communicate to detrusor smooth muscle through the
release of acetylcholine (ACh). In other tissues, such as the vas deferens and mesenteric
arterial circulation, the primary autonomic influence is sympathetic, and norepinephrine
(NE) is the predominant neurotransmitter. NE and ACh act on smooth muscle cells through
Gq-coupled α-adrenergic and muscarinic receptors, respectively, which signal through
PLC to elevate diacylglycerol and inositol trisphosphate (IP3), which in turn, activate
PKC and IP3 receptors (IP3Rs) in the SR. IP3-mediated Ca2+ release from the SR of
vascular smooth muscle cells gives rise to Ca2+ waves (Iino and Tsukioka, 1994; Jaggar
and Nelson, 2000; Wray et al., 2005; Kim et al., 2008), which are propagating elevations
in Ca2+ that are thought to contribute to vascular smooth muscle contraction (Mauban
et al., 2001; Zang et al., 2006). The consequences of G protein–coupled signaling
events manifest after a characteristic lag, reflecting the temporal dynamics of multiple
sequential and parallel molecular linkages.
Although NE and ACh are the prototypical transmitters released by autonomic nerves,
it has long been known that ATP is coreleased with NE at sympathetic nerve–muscular
junctions and with ACh at parasympathetic nerve–muscular junctions. Coreleased ATP
acts on P2X receptor channels in the plasma membrane of smooth muscle cells. Because
P2X receptors are ion channels, once activated, their effects are experienced almost
immediately by the cell. This rapid time course is in contrast to the more delayed
influence of the G protein–coupled adrenergic and muscarinic receptors.
P2X receptors represent a family of seven receptors (P2X1–7) that belong to the transmitter-gated
ion channel superfamily, which also includes nicotinic-like receptors and glutamate-like
receptors (for review see Khakh, 2001). Each P2X receptor subunit possesses intracellular
N and C termini and two membrane-spanning domains linked by a large extracellular
domain (for review see Khakh, 2001; North, 2002). P2X receptors are thought to consist
of three subunits (Aschrafi et al., 2004), which is also the simplest stoichiometry
among ionotropic receptors. At least three ATP molecules bind to the extracellular
domain of P2X channels (Jiang et al., 2003). Upon binding ATP, P2X receptors undergo
conformational changes that result in the opening of the pore within milliseconds,
although the underlying molecular details have not yet been elucidated.
P2X receptors are nonselective cation channels that exhibit a permeability to Ca2+
approximately equal to that of sodium (Na+) (Schneider et al., 1991). Thus, activation
of P2X receptors by ATP released at nerve–muscle junctions causes a rapid local influx
of Na+ and Ca2+ (Lamont and Wier, 2002; Lamont et al., 2006). Although most of the
excitatory junction current (EJC) associated with P2X activation is carried by the
more abundant (∼70-fold) Na+ ions, the influx of Ca2+ is quite substantial. In fact,
the fractional Ca2+ currents mediated by the rat (∼12.4%) and human (∼11%) P2X1 isoforms
are not significantly different from that of the NMDA channel (∼14%) (Egan and Khakh,
2004), long considered the gold standard for high-level, ligand-gated Ca2+ entry.
The current mediated by Na+ and Ca2+ influx creates an excitatory junction potential
(EJP) that contributes directly to the increase in postjunctional excitability associated
with autonomic stimulation.
The P2X1 receptor is the predominant P2X receptor isoform expressed in smooth muscle.
It was originally cloned from the vas deferens (Valera et al., 1994), and immunocytochemical
studies in mice have shown that P2X1 expression in the urinary bladder is restricted
to detrusor smooth muscle (Vial and Evans, 2000). The most compelling evidence for
the prominence of the P2X1 isoform in smooth muscle comes from studies using P2X1
receptor knockout (P2X1R-KO) mice. These studies have shown that ATP-evoked EJCs and
EJPs are absent in the vas deferens from P2X1R-KO mice (Mulryan et al., 2000). Similarly,
these mice lack nerve-evoked purinergic contractile responses in bladders (Vial and
Evans, 2000) and mesenteric arteries (Vial and Evans, 2002; Lamont et al., 2006).
Nerve-evoked elementary purinergic Ca2+ transients: NCTs, jCaTs, and NEPCaTs
As first demonstrated by Brain et al. (2002), the postjunctional action of ATP can
be detected optically in the form of discrete, focal Ca2+ increases in smooth muscle
cells. Using confocal microscopy and a mouse vas deferens preparation in which both
smooth muscle and nerve varicosities are loaded with the Ca2+ indicator dye Oregon
Green 488 BAPTA-1, these authors found that nerve stimulation evokes intermittent
Ca2+ transients at tightly clustered sites immediately adjacent to nerve varicosities.
These events, termed neuroeffector Ca2+ transients (NCTs), are temporally linked to
the stimulating impulse (average delay, ∼6 ms) and are preceded by an increase in
Ca2+ in the adjacent nerve varicosity. NCTs are abolished by persistent exposure to
the P2X agonist/desensitizing agent α,β-methylene ATP (α,β-meATP) and unaffected by
inhibition of voltage-dependent Ca2+ channels (VDCCs), α1-adrenergic receptors, or
IP3Rs (Brain et al., 2003), establishing their likely identity as Ca2+ influx mediated
by ATP-activated P2X receptors.
Shortly thereafter, Wier and colleagues reported similar spatially localized Ca2+
transients in vascular smooth muscle cells of pressurized mesenteric arteries (Lamont
and Wier, 2002). The authors termed these events junctional Ca2+ transients (jCaTs).
Using the Ca2+-binding dye fluo-4 and fluorescence confocal microscopy, these authors
showed that jCaTs are largely unaffected by ryanodine, which abolishes RYR-mediated
Ca2+ sparks. Instead, they are blocked by the nonselective P2X receptor antagonist
suramin, transiently induced by the application of the P2X receptor agonist (desensitizing
agent) α,β-meATP, and absent in P2X1-KO mice, confirming that these events are records
of Ca2+ influx through postjunctional P2X1 receptors (Lamont and Wier, 2002; Lamont
et al., 2006). jCaTs induced by electrical field stimulation of associated nerves
exhibit a close temporal relationship to the stimulus (latency, generally <3 ms).
jCaTs also occur spontaneously, reflecting spontaneous neurotransmitter release.
Work in our laboratory has shown that both spontaneous and nerve-evoked elementary
purinergic Ca2+ transients (NEPCaTs) can also been detected in the urinary bladder.
These events are blocked by suramin and desensitization with α,β-meATP in rat urinary
bladder smooth muscle, and are absent in P2X1R-KO mice (Heppner et al., 2009), showing
that they reflect Ca2+ entry through P2X1 receptor channels. They are also unaffected
by inhibitors of IP3Rs (2-APB), RYRs (ryanodine), or VDCCs (dihydropyridines) (Heppner
et al., 2005), confirming that they are distinct from Ca2+ “puffs,” Ca2+ sparks (Pérez
et al., 1999; Jaggar et al., 2000), and the VDCC-mediated Ca2+ sparklets described
by Santana and Navedo (2009) (see also Table I). In mouse bladders, spontaneous Ca2+
transients are coincident with spontaneous EJPs, and their magnitudes are correlated
(Young et al., 2008), clearly linking these optical events with long-studied, postjunctional
electrical events.
Table I.
Comparison of local Ca2+ transients in smooth muscle
Parameter
Purinergic Ca2+ transients
Ca2+ sparkd
e
Ca2+ pufff
Ca2+ sparkletg
Vas deferens (NCT)a
Mesenteric artery(jCaT)b
Urinary bladder(NEPCaT)c
Duration (t1/2) (ms)
120h; 280i
∼145
∼112
∼56
∼375
(τ) 23; 104
Area (µm2)
∼12
∼25
∼14
∼13.6
2–4
∼0.8
Amplitude (F/F0)
n/d
∼2.8
∼2.0
∼2.0
n/a
n/a
Amplitude (nM)
n/d
n/a
n/a
100–200
50–500
38
Latency (ms)
∼6
<3
8–16
n/a
n/a
n/a
a
Brain et al., 2002.
b
Lamont and Wier, 2002.
c
Heppner et al., 2005.
d
Perez et al., 1999.
e
Jaggar et al., 2000.
f
Ledoux et al., 2008.
g
Santana and Naveda, 2009.
h
Line scan.
i
xy scan.
The kinetic properties of purinergic Ca2+ transients identified in vas deferens (NCTs),
mesenteric arteries (jCaTs), and urinary bladder (NEPCaTs) are similar to one another
and are clearly distinct from those of other focal Ca2+ transients (Table I). The
spatial spread and duration (t1/2) of these events in the bladder are 14 µm2 and ∼112
ms, respectively, and the corresponding values for mesenteric artery jCaTs are ∼20
µm2 and 145 ms. Using line scanning to analyze the kinetics of NCTs, Brain et al.
(2002) showed that NCTs measured in mouse vas deferens have a spatial spread of ∼12
µm2 and decay with a first-order time constant (t1/2) of ∼120 ms. The decay time constant
obtained by xy scanning is much larger (∼280 ms), a difference that was attributed
to the contribution of cytoplasmic diffusion of Ca2+ near the site of entry.
The kinetic properties of spontaneous and evoked purinergic transients are the same,
suggesting that these events are caused by the quantal release of ATP. This is consistent
with earlier evidence that quantal release of ATP is responsible for EJPs and/or EJCs
in femoral and mesenteric arteries, rat tail artery, and vas deferens (for review
see Stjärne and Stjärne, 1995). The low probability, highly intermittent quality of
the events recorded in these electrophysiological studies conforms well with the predictions
of the “intermittent model” developed to describe NE release from sympathetic nerves,
which posits that a single vesicle in ∼1% of all varicosities releases its entire
content in response to a nerve impulse (Stjärne and Stjärne, 1995, and references
therein).
Exploiting this logic, Cunnane and colleagues have used NCTs as a means to detect
“packeted release” of ATP from nerve terminals (Brain et al., 2002; Young et al.,
2007; Brain, 2009). Their results based on electrophysiological measurements in single
smooth muscle cells showed that the amplitude distribution of spontaneous EJPs is
skewed, suggesting a broad distribution of spontaneously released neurotransmitter
packet size (Young et al., 2007). Although bulk changes in ATP release can be monitored
electrophysiologically as EJPs (or EJCs), this approach is less suitable for mapping
quantal transmitter release because smooth muscle cells are large and electrically
coupled, making it difficult to determine whether the recorded event originates in
the cell being recorded (and if so, where), or is caused by release events that occur
at some distance removed from the recording site. Optically measuring Ca2+, released
focally by ATP-activated P2X1Rs, overcomes these limitations, allowing more accurate
spatial mapping of ATP release sites. And because ATP-induced, P2X receptor–mediated
Ca2+ influx is very rapid, optical mapping also provides fine temporal resolution
of the underlying transmitter release events. jCaTs in mesenteric arteries (Lamont
and Wier, 2002) and NEPCaTs in urinary bladder (Heppner et al., 2005) have also been
used to optically map ATP release by sympathetic and parasympathetic nerves, respectively.
In addition to spatial and temporal mapping of ATP release events, NCTs can provide
information about coreleased transmitters and their potential local modulation of
transmitter release probability. One example of this is using NCTs to monitor the
prejunctional autoinhibitory effects of neurally released transmitters. In this context,
the frequency of nerve-evoked NCTs was shown to increase in the presence of the α2-adrenoceptor
inhibitor yohimbine (Brain et al., 2002), providing evidence that coreleased NE acts
through prejunctional α2-adrenoceptors to reduce nerve terminal Ca2+ concentration
and decrease the probability of exocytosis (Brain et al., 2002; Brain, 2009). In a
similar vein, potential off-target effects of pharmacological agents on prejunctional
targets can be inferred from changes in the frequency of purinergic Ca2+ transients
upon the application of such agents, a strategy we have used in studies on the urinary
bladder and mesenteric arteries (unpublished data).
To the extent that release of different transmitters is coupled (i.e., not differentially
regulated), detection of local purinergic Ca2+ transients could provide the means
to optically map nerve activity generally. Whether ATP and NE in sympathetic nerve
terminals are stored and/or released together has been extensively studied by Stjärne
and colleagues. This seemingly straightforward question is deceptively difficult to
answer, especially given the available experimental tools. Early reports from this
group based on electrochemical and electrophysiological studies in rat tail arteries
suggested that ATP and NE are indeed released in parallel by nerve stimulation, with
apparent deviations from this conclusion likely reflecting differences in clearance
rates (for review see Stjärne and Stjärne, 1995). The use of a paired-pulse stimulus
paradigm provided support for this interpretation, showing that the dramatic depression
of paired-pulse transmitter release caused by K+ channel block has similar effects
on NE oxidation currents and ATP-mediated EJCs, and purinergic and adrenergic contractile
responses (Msghina et al., 1998). However, more recent work by these researchers presents
a more nuanced picture. The results of these studies suggest that ATP and NE are stored
in separate small vesicles that are released in parallel upon low frequency stimulation
(<2 Hz), but above this frequency show increasingly nonparallel release (Stjärne,
2001).
Implications of local Ca2+ signaling: Ca2+-signaling networks
Individual purinergic Ca2+ transients have the potential to signal locally to modulate
Ca2+-sensitive processes (Fig. 1). Although this is largely unexplored territory,
some features of such local signaling networks can be discerned from published reports,
and it is possible to speculate about others.
Figure 1.
Local elementary purinergic-induced Ca2+ transients and possible local Ca2+ signaling
networks. ATP released from a nerve varicosity activates smooth muscle P2X1Rs, which
then allow influx of Na+ and Ca2+ ions. Ca2+ influx can induce CICR from RYRs (Brain
et al., 2003) and, in theory, also from IP3Rs. Local influx of Ca2+ may also lead
to activation of NFAT (via calcineurin) or Ca2+-dependent K+ (KCa) channels. Finally,
membrane depolarization caused by Na+ and Ca2+ influx through P2X1Rs would also activate
voltage-dependent ion channels, such as VDCCs or K+ (KV) channels.
Action potential trigger or current injection.
A single purinergic Ca2+ transient represents the activation of a cluster of P2X1Rs
by local ATP from a nerve varicosity. This local injection of current could conceivably
trigger an action potential. Indeed, Young et al. (2008) recently demonstrated that
in intact UBSM strips from mice, single NCTs cause spontaneous depolarizations (“sDeps”)
and can trigger “spontaneous” action potentials. These action potentials cause phasic
contractions, which contribute to muscle tone during bladder filling; an increase
in their activity is a hallmark of unstable detrusor and urinary bladder dysfunction.
The observation that purinergic Ca2+ transients can trigger action potentials suggests
that bladder filling is also under local neurogenic control.
In vas deferens, NCTs do not map to action potentials in a simple one-to-one relationship
(Brain et al., 2002). Although not all NCTs elicit an action potential, ∼20% of NCTs
are rapidly (<0.5 s) followed by an action potential. Consistent with this, purinergic
Ca2+ transients in bladder co-occur with large increases in global Ca2+ termed “flashes”
(Heppner et al., 2005). Both purinergic Ca2+ transients and Ca2+ flashes occur spontaneously,
and the frequency of both types of events is increased by nerve stimulation. Ca2+
flashes are associated with tissue contraction and are eliminated by dihydropyridines,
indicating that they are caused by Ca2+ influx through VDCCs during an action potential.
Purinergic Ca2+ transients are unaffected by inhibition of VDCCs, but inhibition of
P2X receptors abrogates Ca2+ flashes, implying that the cationic flux registered by
the optical NCT/jCaT/NEPCaT event lies upstream of the action potential and is responsible
for triggering it. If sufficiently large, the current and associated depolarization
associated with a single purinergic Ca2+ transient is capable of triggering an action
potential. Presumably, the associated membrane potential depolarization responsible
for activating VDCCs is attributable to the much larger influx of Na+ rather than
the optically registered influx of Ca2+, although this has not been directly tested.
Ca2+-induced Ca2+ release (CICR): NCT/jCaT/NEPCaT to RYR/IP3R communication.
Ca2+ influx through a cluster of P2X1Rs may activate nearby RYRs in the SR, which
should contribute to the purinergic Ca2+ transient (Fig. 1). In vas deferens, unlike
mesenteric artery and urinary bladder smooth muscle, inhibition of RYRs with ryanodine
substantially reduces the amplitude of NCTs (∼45%), and activation of RYRs with caffeine
(3 mM) induces a dramatic (16-fold) increase in the frequency of NCTs (Brain et al.,
2003). In addition, the inhibition of SR Ca2+ uptake by Ca2+ SR/ER-ATPase with cyclopiazonic
acid increases the half-life of these events. These results suggest a functional unit
in which Ca2+ influx mediated by P2X1 stimulates CICR from RYRs, which augments the
local P2X1-mediated Ca2+ signal. According to the model proposed by Brain et al. (2003),
the duration of the Ca2+ signal is governed by the summation of these two Ca2+ release
events as well as the rate at which released Ca2+ is sequestered by the SR through
Ca2+ SR/ER-ATPase pump activity. Such a mechanism would be consistent with the larger
spread and longer half-life of NCT/jCaT/NEPCaTs compared with sparks. Ryanodine reduces
jCaT amplitude by a much more modest, but significant, ∼13% in mesenteric arteries
(Lamont and Wier, 2002), and does not appear to affect purinergic Ca2+ transients
in the urinary bladder (Heppner et al., 2005), suggesting that the effect of RYR inhibition
is at least quantitatively different among these different smooth muscle tissues.
Consistent with the lack of CICR in the regulation of spontaneous phasic contractions,
the inhibition of RYRs does not decrease, but instead enhances, the frequency of phasic
contractions in the urinary bladder smooth muscle from the guinea pig (Herrera et
al., 2000). Depending on the relative speed of IP3 production by concurrent activation
of adrenergic or muscarinic receptors, it is also possible that Ca2+ influx through
P2X1Rs could amplify local IP3R activation by IP3 (Fig. 1). This remains to be explored.
NCT/jCaT/NEPCaT to KCa channel communication.
The timing, nature, and proximity of the actions of coreleased ATP and NE/ACh suggest
that local Ca2+ influx through P2X1 receptors might modulate the subsequent effects
of nerve-evoked NE/ACh release on smooth muscle. Nerve-released ATP acts rapidly on
postjunctional P2XRs to cause a rapid influx of Ca2+ (and Na+), and the coreleased
NE and ACh act more slowly on their respective Gq-coupled receptors. One intriguing
possibility that has not yet been experimentally tested is that the influx of Ca2+
associated with a purinergic Ca2+ transient might activate Ca2+-sensitive K+ (KCa)
channels, such as small-conductance SK or large-conductance BK channels (Fig. 1).
Such a mechanism might conceivably account for our observation that the cholinergic
component of parasympathetic nerve-evoked action potentials in mouse urinary bladder
is augmented by inhibition of P2X receptors (with suramin or α,β-meATP, or by genetic
ablation of P2X1Rs) (Heppner et al., 2009). These results imply that ATP-mediated
P2X1R activity normally exerts an inhibitory influence on the subsequent ACh–muscarinic
receptor signaling pathway. If KCa channels are, in fact, activated by purinergic
Ca2+ transients, their activity would be predicted to dampen cholinergic signaling
and limit the duration of the cholinergic action potential through their membrane
hyperpolarizing action.
Interestingly, purinergic signaling may have the opposite effect on NE signaling in
mesenteric arteries, at least in fully pressurized arteries (90 mmHg). Here, the purinergic
component of sympathetic nerve-evoked vasoconstriction is similar in the presence
and absence of the α1-adrenergic receptor antagonist prazosin, but the adrenergic
component is substantially higher in the presence of functional P2X receptors than
it is with P2X receptors blocked with suramin (Rummery et al., 2007). Thus, it appears
that purinergic activity may exert a potentiating effect on adrenergic signaling in
this setting, consistent with a possible postjunctional influence of smooth muscle
P2X receptors.
NCT/jCaT/NEPCaT to transcription factor communication.
Recent evidence from vascular smooth muscle indicates that VDCCs are associated with
a macromolecular complex containing PKC and AKAP150, as well as calcineurin and the
Ca2+-dependent transcription factor NFAT (Navedo et al., 2008, 2010). Using this parallel,
it is possible to speculate that complexes of P2X1Rs with kinases and phosphatases,
including those that regulate NFAT activation, might also be present in postjunctional
smooth muscle cell membranes (Fig. 1). Colocalization of transmitter receptors (adrenergic
and cholinergic) and ion channels (e.g., VDCCs and KCa channels) in membrane microdomains
might add a further level of regulation to such Ca2+-dependent signaling.
Closing thoughts.
Purinergic Ca2+ transients, by whatever name, are likely a common feature of nerve–smooth
muscle junctions, where they may feed into tissue-specific local Ca2+ signaling networks
and potentially modulate a myriad of Ca2+-dependent processes. Most of the potential
connections between NCT/jCaT/NEPCaT-like events and intracellular signal transduction
have not yet been experimentally tested and remain a matter of conjecture. Regardless
of the functional roles that these events prove to play in postsynaptic smooth muscle
cells, they should provide a convenient and sensitive optical readout of neurally
released ATP specifically and, to an as-yet-undetermined extent, of neural activity
generally.
This Perspectives series includes articles by Gordon, Parker and Smith, Xie et al.,
Prosser et al., and Santana and Navedo.