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The Journal of Neuroscience, August 1, 2001, 21(15):5670-5677
P2X3 Knock-Out Mice Reveal a Major Sensory Role for
Urothelially Released ATP
Mila
Vlaskovska1,
Lubomir
Kasakov1,
Weifang
Rong1,
Philippe
Bodin1,
Michelle
Bardini1,
Debra A.
Cockayne2,
Anthony P. D. W.
Ford2, and
Geoffrey
Burnstock1
1 Autonomic Neuroscience Institute, Royal Free and
University College Medical School, London NW3 2PF, United Kingdom, and
2 Roche Bioscience, Palo Alto, California 94304
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ABSTRACT |
The present study explores the possible involvement of a purinergic
mechanism in mechanosensory transduction in the bladder using
P2X3 receptor knock-out
(P2X3 /) and
wild-type control (P2X3+/+) mice.
Immunohistochemistry revealed abundant nerve fibers in a suburothelial
plexus in the mouse bladder that are immunoreactive to
anti-P2X3. P2X3-positive staining was
completely absent in the subepithelial plexus of the
P2X3/ mice,
whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse
bladder-pelvic nerve preparation, we detected a release of ATP
proportional to the extent of bladder distension in both P2X3+/+ and
P2X3/ mice,
although P2X3/
bladder had an increased capacity compared with that of the
P2X3+/+ bladder. The activity of
multifiber pelvic nerve afferents increased progressively during
gradual bladder distension (at a rate of 0.1 ml/min). However, the
bladder afferents from
P2X3/ mice showed
an attenuated response to bladder distension. Mouse bladder afferents
of P2X3+/+, but not
P2X3/, were
rapidly activated by intravesical injections of P2X agonists (ATP or
 , -methylene ATP) and subsequently showed an augmented response to
bladder distension. By contrast, P2X antagonists
[2',3'-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal
5-phosphate 6-azophenyl-2',4'-disulfonic acid] and capsaicin
attenuated distension-induced discharges in bladder afferents. These
data strongly suggest a major sensory role for urothelially released
ATP acting via P2X3 receptors on a subpopulation of pelvic
afferent fibers.
Key words:
ATP; P2X3 receptor; pelvic afferents; immunohistochemistry; capsaicin; knock-out; mouse; urinary bladder
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INTRODUCTION |
The storage and periodic evacuation
of urine by the bladder is regulated by a complex neural control system
that consists of the CNS and the afferent and efferent spinal
nerves. It has been proposed that lumbosacral afferent fibers (pelvic
afferents) of the urinary bladder principally regulate continence and
micturition (Kuru, 1965 ; de Groat and Steers, 1990). These are
small myelinated (A ) and unmyelinated (C)
fibers that convey impulses from bladder wall receptors. Many
investigators believe that myelinated sacral afferent fibers are
primarily involved in the nonpainful micturition reflex (Bahns et al.,
1986; Mallory et al., 1989), whereas unmyelinated sacral afferents are
activated under painful, pathological conditions (Häbler et al.,
1990). However, the underlying signal transduction mechanism for these
afferents is poorly understood.
There is increasing interest in the role of locally released ATP
in the bladder. Extracellular ATP has been reported to mediate excitation of sensory neurons via P2X receptors, which are ligand-gated ion channels. So far, seven P2X subunits
(P2X1-7) have been cloned, and six of them have
been identified on sensory neurons (Collo et al., 1996; Ralevic and
Burnstock, 1998). Of particular interest is the selective expression of
P2X3 subunits on small-diameter sensory neurons
(Chen et al., 1995; Bradbury et al., 1998) and activation by P2X
agonists of afferent fibers from a variety of tissues (Bland-Ward and
Humphrey, 1997; Cook et al., 1997; Dowd et al., 1998; Hamilton et
al., 2000; Rong et al., 2000). Burnstock (1999) recently put forward a
hypothesis about purinergic mechanosensory transduction that proposed
that in hollow organs, including the ureter and bladder, distension
causes release of ATP from epithelial cells lining these organs and
that ATP can then activate P2X3 receptors on
subepithelial sensory nerve terminals to evoke neural discharge. This
is consistent with the studies of Ferguson et al. (1997), who
demonstrated release of ATP from the serosal epithelium of rabbit
bladder in response to stretch, as well as those of Namasivayam et al.
(1999) who showed using an in vitro rat bladder preparation
that prolonged exposure to a desensitizing dose of ,-methylene
ATP (,-meATP) significantly reduced the activity of
mechanosensitive pelvic nerve afferents in response to distension. More
recently, Cockayne et al. (2000) demonstrated that mice lacking the
P2X3 receptor gene
(P2X3/)
had marked urinary bladder hyporeflexia with reduced voiding frequency
and increased voiding volume, raising the possibility that
P2X3 receptors are closely involved in
mechanosensory transduction underlying the activation of afferent
fibers during bladder filling.
In the present study, we have further explored the sensory role of
P2X3 receptors in the urinary bladder using
P2X3 receptor knock-out
(P2X3/)
and matching wild-type
(P2X3+/+) mice. We aimed
to (1) localize P2X3 immunoreactivity in mouse urinary bladder; (2) quantify the release of ATP during graded distensions of the mouse bladder; and (3) establish an in
vitro mouse bladder-pelvic nerve preparation and characterize the
sensory nerve response to bladder distension and purinergic agonists.
Preliminary results have been reported previously in abstract form
(Vlaskovska et al., 2000).
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MATERIALS AND METHODS |
Animals
Four- to six-month-old male
P2X3/
(27.8 ± 0.4 gm) and matching
P2X3+/+ (30.8 ± 1.1 gm) mice were supplied by Roche Bioscience (Palo Alto, CA). Details
about the generation of these mice have been described by Cockayne et
al. (2000). The animals were kept and handled according to the
regulations of the United Kingdom for the use of transgenic animals.
The mice were killed by exposure to rising concentrations of
CO2 gas.
Immunohistochemistry
P2X3 and Protein Gene Product
9.5 double labeling. The urinary bladder was
dissected, and the trigone zone of the bladder was embedded in OCT
compound (BDH/Merck, Leicester, UK) and frozen in isopentane precooled
in liquid nitrogen. The tissues were sectioned at 12 µm using a
Reichert Jung CM1800 cryostat, collected on gelatin-coated slides, and
air-dried at room temperature. The slides were stored at  20°C and
allowed to return to room temperature for at least 10 min before use.
The sections were immersion-fixed in 4% formaldehyde in 0.1 M phosphate buffer for 2 min. Endogenous
peroxidase was blocked by 10 min incubation in 50% methanol containing
0.4% hydrogen peroxide. Nonspecific protein binding sites were blocked
by a 20 min incubation with 10% normal horse serum (NHS) in PBS
containing 0.05% Merthiolate (Sigma, Poole, UK). Sections were
incubated at room temperature overnight with P2X3
antibody (diluted to 1 µg/ml with 10% NHS). The antibody was raised
in New Zealand rabbits against a synthetic peptide corresponding to the
C terminus of the cloned rat P2X3 receptors
(amino acid fragment 383-397; Oglesby et al., 1999). After this
incubation all washes were performed using PBS containing 0.05% Tween
20 (Sigma). The secondary antibody was a biotinylated donkey
anti-rabbit IgG (Jackson ImmunoResearch, Luton, UK) used at 1:500 for 1 hr, followed by incubation with ExtrAvidin peroxidase (Sigma, Dorset,
UK) diluted 1:1500 for 30 min. The tyramide signal amplification kit
(NEN Life Science Products, Boston, MA) was applied for 8 min, followed
by incubation with Streptavidin fluorescein (Amersham Pharmacia
Biotech, Bucks, UK) diluted 1:100 for 10 min. Slices were
further incubated overnight with the axon marker Protein Gene Product
9.5 (PGP 9.5; UltraClone Ltd., Rossiters Farmhouse Wellow, UK) diluted
1:1000 in PBS containing bovine serum albumin (BSA), lysine,
Triton X-100, and sodium azide. The secondary antibody, donkey
anti-rabbit Cy3 (Jackson ImmunoResearch) was used at a dilution of
1:400 for 1 hr. The slices were observed under a Zeiss Axioplan
microscope (Jena, Germany) at an excitation of 520 nm (for
P2X3 staining) and 570 nm (for PGP 9.5), and
images were captured by a digital camera (Leica, Germany).
Confocal microscopy. The urothelium of the isolated urinary
bladder was carefully stripped off from the smooth muscles and stretched as a whole-mount preparation with inner surface upward on a
Sylgard plate. The tissue was fixed in 4% formaldehyde at room
temperature for 1 hr and then incubated with 10% NHS to block nonspecific binding. Each preparation was incubated overnight with one
of the following antibodies: anti-P2X3 (5 µg/ml), anti-vanilloid receptor 1 (VR1) (1:2000; Chemicon, Temecula,
CA), and anti-CGRP, all diluted in PBS containing BSA, lysine,
Triton X-100, and sodium azide (all from Sigma). The secondary
antibody, biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch)
diluted 1:500 in 1% NHS was applied for 1 hr followed by 1 hr
incubation with streptavidin FITC (Amersham Pharmacia Biotech) diluted
1:100. The tissue was then incubated in Pontamine Sky Blue for 5 min to
reduce autofluorescence. The tissue was observed under a confocal
microscope at an excitation of 488 nm, and images were captured by a
digital camera.
In vitro urinary tract preparation and measurement
of ATP release
The whole urinary tract attached to the lower vertebrae and
surrounding tissues was quickly isolated en bloc and placed
in a chamber where it was continuously superfused with oxygenated (5%
CO2 and 95% O2) Krebs'
solution (contents in mM: NaCl 120; KCl 5.9;
NaH2PO4 1.2;
MgSO4 1.2; NaHCO3 15.4;
CaCl2 2.5; and glucose 11.5). The chamber
temperature was kept at ~26°C. A 25 gauge needle was inserted into
the lumen of the urinary bladder and was connected to a syringe-type
infusion pump (sp210iw; World Precision Instruments, Sarasota,
FL), a pressure transducer (NL108T2; Digitimer, Hertfordshire, UK), and a 100 µl Hamilton syringe via an Omnifit. This enabled infusion and withdrawal of medium (Krebs' solutions) at a constant rate (0.1 ml/min), recording of intraluminal pressure, and intravesical injection of drugs. The preparation was allowed to stabilize for at
least 60 min.
ATP released from urothelial cells was measured in 100 µl samples
taken from the intravesical medium. Before each distension, four or
five samples were collected from wash-out medium at 5 min intervals
(baseline ATP levels). The bladder was distended to intraluminal
pressures of 7.5, 15, 20, and 25 mmHg for 2 min at random. ATP levels
were quantified (in picomoles per milliliter) as previously
described (Bodin and Burnstock, 1996). Briefly 50 µl aliquots were
taken from each sample and pipetted in duplicates onto a multi-well
non-phosphorescent microplate. This was placed in a
luminometer (Lucyl; Anthos Labtec, Salzburg,
Austria) and processed automatically by injection of 100 µl
luciferin-luciferase reagent (Bio-Orbit, Turku, Finland) into
each well. The light emission was counted for 10 sec, and ATP
concentration was extrapolated from a calibration curve constructed
using standard ATP solutions prepared in Krebs' solution (pH adjusted
to 6 by 0.1 N HCl). The results are presented as mean ± SEM. Data
were compared by Student's t test, and differences
considered statistically significant at p < 0.05.
Electrophysiology
The mouse urinary tract was dissected and perfused as stated
above. With the aid of a dissecting microscope, a branch of the pelvic
nerve arising from the urinary bladder was dissected. The nerve
activity was recorded with a suction glass electrode (tip diameter,
50-100 µm) connected to a Neurolog head stage (NL 100; Digitimer)
and an AC amplifier (NL 104; Digitimer). Signals were amplified
(10,000×), filtered (band-pass 200-4000 Hz), and relayed to a D310
spike processor (Digitimer) that discriminates neural impulses from
noise with a manually set amplitude and polarity window. The nerve
activity and the intraluminal pressure were recorded on tape and a
computer with a power 1401 analog-to-digital interface and
Spike2 software (Cambridge Electronic Design, Cambridge, UK). The nerve
activity was counted and plotted as rate histogram.
In pilot studies, the characteristics of neuronal response to bladder
distension and the stability of the model was tested by repeatedly
filling the bladder with Krebs' solution at a rate of 0.1 ml/min and
at intervals of 10-15 min. Consistent neuronal response to distension
could be obtained for as long as 10 hr. To quantify the changes in
nerve activity in response to bladder distension, each preparation was
first distended at a rate of 0.1 ml/min until the nerve activity
reached a plateau (control maximal activity), and this was repeated
three times (interval between distension = 10 min). The average
firing rate in every 15 sec period (i.e., for every 25 µl of injected
volume) was counted, expressed as a percentage of the maximal activity
(normalized nerve activity), and plotted against the volume of medium
in the bladder. The effects of P2X agonists on the pelvic afferents and their response to distension were tested by injecting 0.1 ml solutions containing the agonists as a bolus into the empty bladder and distending the bladder 10 min thereafter. The effects of P2X
antagonists and capsaicin were examined using a similar protocol.
Chemicals
ATP (disodium salt), ,-meATP (lithium salt,), and
pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid
(PPADS) (all obtained from Sigma, St. Louis, MO) were diluted to
final concentrations in Krebs' solution, and the pH was adjusted to
7.4. Capsaicin (Tocris Cookson, Bristol, UK) was dissolved in DMSO as a
10 mM stock solution and diluted to final concentrations in
Krebs' solution before use. All inorganic salts were purchased from
BDH Laboratory Supplies (Poole, UK).
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RESULTS |
Immunohistochemical characterization of bladder
sensory afferents
As illustrated in Figure 1,
A and D, intense immunofluorescent staining for
P2X3 subunits was found in nerve bundles between the smooth muscle layer of the bladder wall in
P2X3+/+, but not in
P2X3/
mice. However, staining for PGP 9.5, an axon marker, was found in the
bladder from both P2X3+/+
and
P2X3/
mice (Fig. 1B,E). The staining for
P2X3 subunits coexisted with that of PGP 9.5 in
the P2X3+/+, but not in
the
P2X3/
mouse bladder (Fig. 1C,F).
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Figure 1.
Double staining for P2X3 and PGP 9.5 in the mouse urinary bladder. Top and bottom
panels are sections of the bladder (with all layers of the
bladder wall) from P2X3 wild-type
(P2X3+/+) and P2X3 knock-out
mice (P2X3/),
respectively. A, D, Immunofluorescence
(green) for P2X3 receptor. Note that
the nerve bundle within the smooth muscle coat is densely stained for
the P2X3 subunit. B, E, immunofluorescence
(red) for PGP 9.5, an axon marker. C,
F, Co-staining for P2X3 and PGP 9.5. L, Luminal side of the bladder wall; S,
serosal side of the bladder wall; M, smooth muscle.
Scale bar, 100 µm.
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Whole-mount bladder urothelial preparations examined with confocal
microscopy revealed a dense suburothelial plexus of CGRP-immunoreactive nerve fibers in P2X3+/+
as well as
P2X3/
mice (Fig. 2A,D).
Similarly, VR1-immunoreactive fibers (Fig. 2B,E) were
also found in the suburothelial plexus from both
P2X3+/+ and
P2X3/
mice. Although numerous P2X3-positive fibers were
found in the suburothelial plexus and in those projections between
urothelial cells in the bladder of
P2X3+/+ mice (Fig.
2C), none were seen in the suburothelial plexus of P2X3/
mice (Fig. 2F).
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Figure 2.
Immunostaining of the subepithelial sensory nerve
plexus in the mouse bladder. Each panel represents the inner surface of
a small piece of urothelium (clear of smooth muscles).
Top and bottom panels are urothelium from
P2X3 wild-type
(P2X3+/+) and
P2X3 knock-out mice
(P2X3/),
respectively. A, D, Immunofluorescence
for CGRP; B, E, Immunofluorescence for VR1; C,
F, Immunofluorescence for P2X3 subunits. Scale bar,
100 µm.
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Distension-induced ATP release from the urothelium
The bladder was distended by intravesical infusion of Krebs'
solution at a constant rate (0.1 ml/min) until the intraluminal pressure reached a certain level. It was noticed that the bladders of
P2X3/
mice typically had a larger capacity than those of
P2X3+/+ mice such that
the same levels of intravesical pressure could be achieved after
infusion of larger volumes of medium. In bladders from
P2X3+/+ mice, 7.5 mmHg
was achieved with 110 ± 5 µl medium, 15 mmHg with 215 ± 10 µl, 20 mmHg with 320 ± 20 µl, and 25 mmHg with 430 ± 15 µl. In bladders from
P2X3/
mice, the same intravesical pressure was produced by infusion of
135 ± 15, 245 ± 15, 360 ± 20, and 480 ± 25 µl, respectively. The basal release of ATP was measured in the
samples taken from the medium during every predistension period. The
baseline levels of ATP were similarly low during all predistension
periods in both P2X3+/+
and
P2X3/
mice, being 0.5 ± 0.1 and 0.6 ± 0.1 pmol/ml, respectively.
The threshold for distension-evoked ATP release was 3.5-4.0 mmHg, which corresponded to intravesical volume of 60-70 µl for the P2X3+/+ mouse bladder.
With increasing intravesical volume (and pressure), ATP level increased
progressively. Thus, distension to an intravesical pressure of 7.5 mmHg
produced a several-fold incremental increase of ATP over the baseline
level. The plateau of intravesical ATP levels was reached at an
intravesical pressure of 20 mmHg, which corresponded to intravesical
volume of 300 ± 20 µl. Distension of
P2X3/
bladder produced an identical pattern of ATP release. The results of
this series of experiments are summarized in Table
1.
Activity of pelvic afferent fibers evoked by distension of the
bladder and P2X agonists
The pelvic afferent fibers from the urinary bladder of
P2X3+/+ and
P2X3/
mice both had low background activity (0-10 spikes/sec). Examples of
distension-induced changes in intravesical pressure and discharges in
pelvic afferents in bladder-pelvic nerve preparations from P2X3+/+ and
P2X3/
mice are illustrated in Figure 3. Figure
4 summarizes the data from this series of
experiments. In most preparations, biphasic activation of afferent
fibers in association with increases in intravesical pressure was
observed with an initial slow rise followed by a later rapid increase
in pressure and nerve activity. However, the pelvic afferents of
P2X3/
mice were less sensitive to bladder distension than those of P2X3+/+ mice (Fig. 3,
compare A, B). In the
P2X3+/+ mice, nerve
activity started to increase when the bladder was distended to a volume
of 115.6 ± 11.6 µl (intravesical pressure of 5.7 ± 1.0 mmHg; n = 8), whereas in the
P2X3/
mice, the threshold for significant increase in nerve activity was
276.7 ± 24.0 µl (intravesical pressure of 10.2 ± 1.6 mmHg; n = 6) (Fig. 4). In
P2X3+/+ mice, nerve
activity reached a peak when the bladder was distended to a volume of
472.6 ± 51.8 µl (intravesical pressure of 36.1 ± 4.7 mmHg; n = 8), whereas in the
P2X3/
mice, peak nerve activity was achieved at significantly larger intravesical volumes (729.1 ± 57.3 µl, corresponding to a
pressure of 40.5 ± 6.1 mmHg; n = 6) (Fig. 4).
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Figure 3.
Distension-induced discharges in mouse bladder
afferents. A, Wild-type mice
(P2X3+/+).
B, P2X3 knock-out mice
(P2X3/).
The bladder was distended at a constant rate of 0.1 ml/min. Note the
much delayed activation of the bladder afferents in the
P2X3/
preparation.
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Figure 4.
A summary of the alterations in intravesical
pressure and bladder afferent activity during distension of the
bladder. The nerve activity was normalized and expressed as the
percentage of maximal activity. Data representative of an
n = 8 for P2X3-wild type mice
(P2X3+/+) and an
n = 6 for P2X3-knock-out mice
(P2X3/).
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The effects of P2X agonists (,-meATP and ATP) and capsaicin on
the pelvic afferents of
P2X3+/+ mice
(n = 7) and
P2X3/
mice (n = 5) are shown in Figure
5. The agonists were injected into the
empty bladder as a 100 µl bolus. Injecting 100 µl of vehicle into
the bladder did not evoke significant changes in intravesical pressure
and nerve activity. In the
P2X3+/+ mice (Fig.
5A1), ,-meATP (100-300 µM)
induced a rapid increase in the discharge of the pelvic afferents, and
the response lasted for 5-10 min. Similarly, ATP (Fig. 5B1)
induced a rapid excitation of the pelvic afferents, but the minimal
concentration of ATP (1 mM) required to evoke a
significant neural response was ~10-fold higher than that of
,-meATP (100 µM). Neither ATP nor
,-meATP had any significant effect on the activity of the pelvic
nerve of the
P2X3/
mice (Fig. 5A2,B2). However, capsaicin (30 µM) induced significant afferent activation in
both P2X3+/+ and
P2X3/
preparations. All these agonists also produced similar small increases
in intravesical pressure in both
P2X3+/+ (Fig.
5C1) and
P2X3/
(Fig. 5C2) preparations.
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Figure 5.
Responses of multifiber bladder afferents to
intravesical administration of P2X agonists and capsaicin.
Left, Wild-type control mice
(P2X3+/+). Right,
P2X3 knock-out mice
(P2X3/). Agonists
(,-meATP, 100 µM; ATP, 1 mM; capsaicin,
30 µM) were injected into the empty bladder in a volume
of 100 µl. All recordings on the left column are from
same P2X3+/+ mouse, and those on the
right column are from same
P2X3/
mouse.
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The effects of ,-meATP and capsaicin agonists on the afferent
nerve response to distension were further examined by distending the
bladder 10 min after the administration of the agonists. As demonstrated in Figures 6 and
7, ,-meATP did not affect the pressure changes during distension. However, in
P2X3+/+ preparations the
afferent nerve response to distension was greatly potentiated by
preinfusion of agonist, in that nerve activity increased faster and
reached a greater peak level than that observed with vehicle alone
(Figs. 6, 7). Recovery of the distension-induced afferent nerve
responses was achieved after washout of the agonist. The potentiation
was not seen in the
P2X3/
preparation (n = 2). In contrast, after administration
of capsaicin, the afferent nerve response to bladder distension was
significantly attenuated with peak activity reduced to 34.7 ± 5.8% (n = 5; p < 0.01) of the
control.
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Figure 6.
Effects of ,-meATP on the distension-induced
changes in multifiber bladder afferent activity in a P2X3
wild-type (P2X3+/+) mouse.
A and B are sequential recordings in same
preparation. In A, an injection of vehicle (Krebs', 0.1 ml) was followed by bladder distension (0.1 ml/min). In
B, distension (0.1 ml/min) was preceded by an injection
of ,-meATP (0.3 mM, 0.1 ml), which itself elicited
afferent discharges and potentiated distension-induced
discharges.
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Figure 7.
A summary of the effects of ,-meATP on
distension-induced changes in bladder afferent activity in
P2X3 wild-type (P2X3+/+)
mice. The experimental protocol was the same as shown in Figure 6. The
nerve activity was normalized and expressed as a percentage of the
maximal firing rate reached during the control distension with vehicle.
Data were pooled from six P2X3+/+ mouse
bladder-nerve preparations.
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The neural response to P2X agonists in the
P2X3+/+ control mice
could be blocked by the P2X antagonists PPADS (n = 4)
and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)
(n = 4). This is exemplified in Figure
8, which illustrates the attenuation in
nerve activity induced by intravesical injection of ,-meATP
after, but not before, preincubating the preparation intravesically
with TNP-ATP (Fig. 8, compare A, B). Both PPADS and TNP-ATP
were also able to reduce the neural response to bladder distension with
a representative recording shown in Figure
9. Distension-induced peak activity was
reduced after pretreatment with TNP-ATP (30 µM)
and PPADS (300 µM) to 65.3 ± 18.4%
(n = 4; p < 0.05) and 47.1 ± 17.3% (n = 4; p < 0.01) of the
vehicle control, respectively.
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Figure 8.
Effects of TNP-ATP on bladder afferent
responses to ,-meATP. A, An intravesical
injection of ,-meATP (100 µM, 0.1 ml) induced
significant discharges in the bladder afferents. B, The
preparation was incubated intravesically with the P2X antagonist
TNP-ATP (30 µM, 0.1 ml) for 10 min; a subsequent
injection of ,-meATP (100 µM, 0.1 ml) after removal
of the TNP-ATP produced little change in nerve activity.
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Figure 9.
Effects of PPADS on the distension induced changes
in bladder afferent activity in a P2X3 wild-type
(P2X3+/+) mouse bladder-nerve
preparation. A, Nerve activity in control situation.
B, Nerve activity after application of PPADS (300 µM).
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DISCUSSION |
Morphology of the sensory innervation of the bladder
The major function of the urinary bladder is to store and evacuate
urine. The significance of the sensory innervation of the bladder is to
detect the filling of the bladder and convey this information to the
CNS to evoke the micturition reflex. The innocuous sensory information
about bladder fullness is transformed to a painful sensation (or
discomfort) when the intravesical pressure reaches a threshold of
30-50 mmHg (Häbler et al., 1990). Two types of afferent fibers
are involved, namely myelinated A - and unmyelinated C-afferent
fibers of the pelvic and the hypogastric nerves (McGuire, 1986), which
comprise low- and high-threshold mechanosensory receptors (Sengupta and
Gebhart, 1994). Detailed immunohistochemical (CGRP) and
immunofluorescent (synaptophysin) studies have revealed dense plexuses
of afferent axons in a close apposition to the urothelium (Gabella and
Davis, 1998). The afferent axons were shown to be associated with four
major targets: the base of the urothelium, the space between urothelial
cells, blood capillaries, and smooth muscle cells. In the mucosa, all
afferent axons are located between urothelial cells or in the
suburothelial plexus adjacent to the basal surface of urothelium.
Immunohistochemical studies have revealed that although the detrusor
muscle expresses P2X1 receptors (Ferguson, 1999;
Lee et al., 2000), P2X3 receptors are present in
the urothelium (Ferguson, 1999) and specifically on afferent nerve
fibers (Cockayne et al., 2000). In agreement with these data, we found
abundant CGRP, VR1, and P2X3
receptor-immunoreactive nerve fibers running between urothelial cells,
in the suburothelial plexus, and perivascularly in the urinary bladder
of P2X3+/+ mouse (Fig.
2). P2X3 immunostaining coexists with staining
for the neuronal marker PGP 9.5 (Fig. 1), indicating the neuronal localization of P2X3 receptors. As expected,
P2X3 receptor immunoreactivity was completely
absent in the bladder of
P2X3/
mice, but the immunoreactivity for CGRP and VR1 seemed unaltered (Fig.
2). The importance of the P2X3-positive afferents
in sensory signal transduction in the bladder is signified by the
findings of Cockayne et al. (2000) that
P2X3-deficient mice display marked urinary
bladder hyporeflexia.
Distension induced release of ATP from the bladder
In a rabbit bladder preparation, Ferguson et al. (1997) recorded
changes in short-circuit currents and transepithelial potential that
were accompanied by a substantial release of ATP from the serosal, but
not luminal side of the urothelium in response to stretch. It was
suggested that urothelium was a sensor for pressure in the bladder,
which released ATP through nonvesicular mechanisms. In the present
study, we detected a significant release of ATP into the lumen during
distension of the mouse urinary bladder. In normal control mice, the
threshold for the release of ATP was 3.5-4.0 mmHg of intravesical
pressure, indicating that innocuous filling of the bladder may evoke
release of ATP. We further demonstrated that distension-induced release
of ATP is preserved in
P2X3/ mice.
Electrical activity of the pelvic nerve fibers from
the bladder
The electrophysiological properties of sensory innervation in the
lower urinary tract have been studied extensively in the rat (Mallory
et al., 1989) and cat in vivo (Bahns et al., 1986; Janig and
Morrison, 1986; Häbler et al., 1990) and more recently in the rat
in vitro (Namasivayam et al., 1998, 1999). The mouse has so
far been a neglected species in the study of neural control of the
urinary tract, and little is known about the functional properties of
afferent nerves from the mouse bladder. However, with more transgenic
mice becoming available, this species is becoming an increasingly
important experimental animal in bridging the information from
molecular biology and systems physiology. In this paper, we describe a
novel in vitro mouse bladder-pelvic nerve preparation.
Using this model, we have, for the first time, examined the
characteristics of the pelvic afferent response to bladder distension.
The response consists of a progressive increase in firing rate in
association with rises in intravesical pressure. Our observations
indicate that this preparation is a relatively simple and reliable
model for investigating sensory signal transduction mechanisms in the
urinary bladder.
Namasivayam et al. (1999) recently reported the use of an in
vitro rat bladder-pelvic nerve preparation to demonstrate that distension induced by an intravesical infusion of a solution containing 10 µM ,-meATP produced slightly greater
responses at first and then significantly attenuated responses in
pelvic afferents. These effects of ,-meATP were attributed to the
activation and subsequent desensitization of P2X receptors located on
afferent terminals on the urinary bladder. In support of this concept,
we have localized P2X3 receptor immunoreactivity
on afferent nerve fibers located in the mouse urinary bladder. Our
electrophysiological observations demonstrate that intravesical
applications of P2X agonists (ATP or ,-meATP) induce a rapid
excitation of bladder afferents in the
P2X3+/+ mice.
Furthermore, ,-meATP greatly potentiated subsequent
distension-induced afferent discharges. This seems to contradict the
observations of Namasivayam et al. (1999). However, this discrepancy
may be attributable to the difference in the concentration and the
speed of delivery of the agonist. Although we injected ,-meATP
(100-300 µM, 0.1 ml) into the bladder as a
bolus, they distended the bladder slowly with ,-meATP (10 µM).
A significant change in electrophysiological properties of bladder
afferents in
P2X3/
mice is their diminished response to P2X agonists (Fig. 5) and their
attenuated response to bladder distension, which was manifested by a
significantly delayed and increased threshold of activation of the
afferents when compared with those from
P2X3+/+ mice (Figs. 3,
4). This strongly indicates that P2X3 subunits on
afferent terminals may play an important part in mechanosensory transduction in the urinary bladder. However, as illustrated in Figure
4,
P2X3/
bladder generates less intravesical pressure for a given distension. On
one hand, this is not unexpected if mechanosensory transduction in the
bladder involves a purinergic component of P2X3
subunits a deficiency of which may lead to increased threshold for
micturition and consequently compensatory increase in bladder capacity.
On the other hand, it is likely that this reduced bladder pressure response (or increased bladder capacity) in the
P2X3/
mice may also partially account for the attenuated nerve response to
bladder distension.
Previous studies indicate that there may be different functional
classes of bladder afferents. A afferents in the cat respond in a
graded manner to distension of the bladder (Janig and Morrison, 1986)
and are activated by noxious stimuli as well. On the other hand, C
fibers in the cat have very high mechanical thresholds and commonly do
not respond to even high levels of intravesical pressure (Häbler
et al., 1990). The activity in some of these afferents, however, is
triggered by chemical irritation of the bladder mucosa (Häbler et
al., 1990) or cold (Fall et al., 1990). Single-unit studies will be
necessary to determine whether a purinergic mechanism preferentially
modulates the sensory transduction in one of the different classes of
bladder afferents.
In other organs, capsaicin has been used to differentiate between
innocuous and nociceptive (A and C) fibers. However, whereas afferents from the bladder consist largely of A and C fibers, both
are activated by capsaicin. In the present study capsaicin was able to
significantly attenuate afferent response to distension and
intravesical administration of P2X agonists. This is consistent with
the immunohistochemical colocalization of P2X3
and VR1 receptors in afferent nerve fibers. Interestingly, rats treated
with capsaicin as neonates were found to excrete less urine
(Holzer-Petsche and Lembeck, 1984). Their bladders were found to have a
larger capacity (5 ml) than normal controls (1 ml), and systemic
capsaicin treatment was able to block the rhythmic contraction of
detrusor muscles in response to distension. Clinically, intravesical
administration of capsaicin has been proven effective in treating
incontinence (Chancellor and de Groat, 1999). Given that P2X agonists
and capsaicin can affect the activity of the same set of afferent
fibers, P2X3 agonists/antagonists may be of
therapeutic use in urological conditions arising from disorders
affecting the sensory mechanisms in the bladder.
In summary, we have demonstrated the presence of
P2X3 receptors on afferent nerve fibers closely
associated with the urothelium and the release of ATP by distension of
the mouse urinary bladder in a novel in vitro mouse
bladder-pelvic nerve preparation. We found that P2X agonists were able
to activate pelvic afferents and potentiate their response to bladder
distension in P2X3+/+,
but not in
P2X3/
mice that showed attenuated pelvic afferent response to bladder distension. These data strongly suggest a major sensory role of urothelially released ATP via activation of P2X3
receptors on a subpopulation of pelvic afferent nerve fibers.
| |
FOOTNOTES |
Received Dec. 28, 2000; revised April 2, 2001; accepted April 17, 2001.
This work was supported by grants from the Wellcome Trust (M.V., L.K.,
W.R.) and the British Heart Foundation (P.B).
Correspondence should be addressed to Dr. Weifang Rong, Autonomic
Neuroscience Institute, Royal Free and University College Medical
School, Rowland Hill Street, London NW3 2PF, UK. E-mail: ucgaron{at}ucl.ac.uk.
| |
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ABSTRACT
INTRODUCTION
