Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Yoshimura, Naoki,
Satoshi Seki, and
William C. de Groat.
Nitric Oxide Modulates Ca2+ Channels in Dorsal Root
Ganglion Neurons Innervating Rat Urinary Bladder.
J. Neurophysiol. 86: 304-311, 2001.
The
effect of a nitric oxide (NO) donor on high-voltage-activated
Ca2+ channel currents
(ICa) was examined using the whole
cell patch-clamp technique in
L6-S1 dorsal root ganglion
(DRG) neurons innervating the urinary bladder. The neurons were labeled
by axonal transport of a fluorescent dye, Fast Blue, injected into the
bladder wall. Approximately 70% of bladder afferent neurons exhibited
tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these
neurons were sensitive to capsaicin, while the remaining neurons had
TTX-sensitive spikes and were insensitive to capsaicin. The peak
current density of nimodipine-sensitive L-type
Ca2+ channels activated by depolarizing pulses (0 mV) from a holding potential of 60 mV was greater in bladder afferent
neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent
neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of
-conotoxin GVIA-sensitive N-type Ca2+
channels was similar (43-45 pA/pF) in both types of neurons. In both
types of neurons, the NO donor,
S-nitroso-N-acetylpenicillamine (SNAP) (500 µM), reversibly reduced (23.4-26.6%) the amplitude of
ICa elicited by depolarizing pulses to
0 mV from a holding potential of
60 mV. SNAP-induced inhibition of
ICa was reduced by 90% in the
presence of
-conotoxin GVIA but was unaffected in the presence of
nimodipine, indicating that NO-induced inhibition of
ICa is mainly confined to N-type
Ca2+ channels. Exposure of the neurons for 30 min
to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 µM), an
inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced
reduction in ICa. Extracellular
application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by
reducing the peak amplitude of ICa
(28.6% of reduction). Action potential configuration and firing
frequency during depolarizing current pulses were not altered by the
application of SNAP (500 µM) in bladder afferent neurons with
TTX-resistant and -sensitive APs. These results indicate that NO acting
via a cGMP signaling pathway can modulate N-type
Ca2+ channels in DRG neurons innervating the
urinary bladder.
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INTRODUCTION |
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Nitric oxide (NO) has been
identified as a transmitter at various sites in the neural pathways
controlling the urogenital organs. In the peripheral nervous system, NO
released by parasympathetic nerves mediates penile erection and
urethral relaxation (Andersson 1993; Andersson
and Persson 1995
); whereas NO released in the spinal cord
appears to facilitate the hyperactivity of the urinary bladder induced
by noxious stimulation of the lower urinary tract (Kakizaki and
de Groat 1996
; Rice 1995
). NO is also
released by afferent nerves and epithelial cells in the urinary bladder
(Birder et al. 1997
, 1998
); however, its function at
these sites is uncertain. Some studies indicate that NO has a
facilitatory effect of afferent activity and may participate in the
initiation of inflammatory responses and the triggering of painful
sensations (Aley et al. 1999
). Chronic irritation of the
urinary bladder increased the expression of neuronal nitric oxide
synthase (nNOS) in bladder afferent neurons (Vizzard et al.
1996
). Up-regulation of this enzyme, which is responsible for
the synthesis of NO, suggests that pathological conditions might
increase the effects of NO.
Other studies indicated that NO might have a depressant effect on
afferent activity. Reduced nitrite levels in the urine of patients with
interstitial cystitis, a painful bladder condition, suggested that
decreased NO release in the bladder might contribute to the disorder
(Smith et al. 1996). Patients treated with
L-arginine to increase the synthesis of NO exhibited some
reduction in symptoms, raising the possibility that NO might have an
antinociceptive action (Smith et al. 1997
). In the rat,
intravesical administration of an NO donor
[S-nitroso-N-acetylpenicillamine (SNAP)]
depressed bladder hyperactivity induced by the administration of a
chemical irritant, cyclophosphamide (CYP) (Ozawa et al.
1999
). Because NO has only minimal relaxing effects on rat
bladder smooth muscle (Andersson 1993
; Andersson
and Persson 1995
), it was suggested that exogenous NO depressed
reflex bladder activity by suppressing the excitability and/or the
release of transmitters from bladder afferent nerves. It is well known
that release of neuropeptides such as substance P from C-fiber bladder
afferents contributes to hyperactivity and inflammatory responses in
the bladder (Lecci et al. 1994
; Maggi et al.
1992
). Previous experiments also revealed that
high-voltage-activated (HVA) Ca2+ channels are
involved in the release of neuropeptides such as substance P
from capsaicin-sensitive bladder and other visceral afferent terminals
(Maggi et al. 1989
, 1990
; Waterman 2000
)
and that NO can modulate various types of ion channels including
Ca2+ channels in the mammalian neurons (Li
et al. 1998
). It is therefore possible that the depressant
effects of NO on bladder hyperactivity are mediated by modulation of
Ca2+ influx in afferent nerve terminals. However,
the cellular mechanisms of action of NO in bladder afferent pathways
have not been studied in detail.
In the present study, we have used dissociated bladder afferent neurons
as a model system to evaluate the actions of NO on bladder sensory
pathways. In a preliminary report, we showed that NO donors such as
sodium nitroprusside and SNAP can suppress HVA Ca2+ channel currents in bladder afferent neurons
of the rat (Ozawa et al. 1999; Yoshimura and de
Groat 1997a
). The present study was conducted to further
evaluate the effects of NO on specific types of HVA
Ca2+ channels in the two subtypes of bladder
afferent neurons. Our results demonstrate that NO can inhibit HVA
Ca2+ channels in A
- and C-fiber bladder
afferent neurons, which were classified according to TTX and capsaicin
sensitivity. This effect was primarily confined to N-type
Ca2+ channels and mediated through a cyclic GMP
(cGMP)-dependent mechanism.
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METHODS |
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Animal preparation
Experiments were performed on adult female Sprague-Dawley rats (150-250 g). Care and handling of animals were in accordance with institutional guidelines and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
As previously described (Yoshimura and de Groat 1997b,
1999
; Yoshimura et al. 1998
), the population of
DRG neurons that innervate the urinary bladder were labeled by
retrograde axonal transport of a fluorescent dye, Fast Blue (4%
wt/vol) (Polyloy, Gross Umstadt, Germany), injected into the wall of
the bladder in halothane-anesthetized animals 7 days prior to the
dissociation. The dye was injected with a 29 G needle at three to six
sites on the dorsal surface of the bladder (5-6 µl per site, total
volume of 20-30 µl). Each injection site was washed with saline to
minimize contamination of adjacent organs with the dye. Particular care
was taken to avoid injections into the lumen, major blood vessels, or
overlying fascial layers to minimize nonspecific labeling due to dye
leakage. No apparent leakage was observed.
Cell dissociation
Freshly dissociated neurons from DRG were prepared from
halothane anesthetized animals (Yoshimura et al. 1996).
Briefly, L6 and S1 DRG were
dissected and then dissociated in a shaking bath for 25 min at 35°C
with 5 ml of Dulbecco's modified Eagle's medium (Sigma) containing
0.3 mg/ml trypsin (Type 3, Sigma), 1 mg/ml collagenase (Type 1, Sigma),
and 0.1 mg/ml deoxyribonuclease (Type 4, Sigma). Trypsin inhibitor
(Type 2a, Sigma) was then added to neutralize the activity of trypsin.
Individual DRG cell bodies were isolated by trituration and then plated
on poly-L-lysine-coated 35-mm petri dishes.
Electrical recording
Dye-labeled primary afferent neurons that innervate the urinary
bladder were identified using an inverted phase contrast microscope (Nikon, Tokyo) with fluorescent attachments (UV-1A filter; excitation wave length, 365 nm). Gigaohm-seal whole cell recordings were performed
at room temperature (20-22°C) on each freshly dissociated labeled
neuron in a culture dish that usually contained two to five labeled
cells among a few hundred unlabeled neurons. The internal solution used
during current-clamp recordings of action potentials contained (in mM)
140 KCl, 1 CaCl2, 2 MgCl2,
9 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 GTP (Tris Salt) adjusted to pH 7.4 with KOH (310 mOsm). Patch electrodes had resistances of 1-4 M when
filled with the internal solution. Neurons were superfused at a flow
rate of 1.5 ml/min with an external solution containing 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES and 10 D-glucose, adjusted to pH 7.4 with NaOH (340 mOsm). All recordings were made with an Axopatch-1D patch-clamp
amplifier (Axon Instruments, Foster City, CA), and data were acquired
and analyzed by pClamp software (Axon Instruments). Cell membrane
capacitances were obtained by reading the value for whole cell input
capacitance neutralization directly from the amplifier. Durations of
action potentials were measured at 50% of the spike amplitude.
Thresholds for action potential activation were determined by injection
of depolarizing current pulses in 20-pA steps. In current-clamp
recordings, data are presented from neurons that exhibited resting
membrane potentials more negative than
40 mV and action potentials
that overshot 0 mV.
After current-clamp recordings, Ca2+ channel
currents conducted by Ba2+ ions were isolated by
switching the external solution containing (in mM) 5 BaCl2, 155 tetraethylammonium (TEA)-Cl, 5 4-aminopyridine, and 10 HEPES adjusted to pH 7.4 with TEA-OH (340 mOsm). Using this external solution, high-threshold inward currents
[mean peak amplitude; 3.5 ± 0.3 (SE) nA,
n = 44 cells] elicited by depolarizing pulses to 0 mV
from a holding potential of 60 mV were almost completely suppressed
by 400 µM CdCl2 with a residual outward current
of 21.6 ± 3.9 pA (n = 11), indicating that
contamination by K+ currents was minimal under
this condition. Cells in which excessive rundown was observed in a
control period ranging up to 5 min were excluded from the experiments.
In voltage-clamp recordings, the filter was set to
3 dB at 2,000 Hz.
Leak currents were subtracted by P/4 pulse protocol and the series
resistance was compensated by 60-80%. All recordings were performed
within 12 h after dissociation.
TTX, nimodipine, and SNAP were applied to neurons by superfusion of the
external solution containing each drug. Capsaicin (1 µM) and
-conotoxin GVIA (3 µM) were directly applied to the cells by
pressure ejection (Picospitzer, General Valve, Fairfield, NJ) through a
glass pipette (10-20 µm tip diameter, 500 ms at 5-10 psi). Inward
shift of holding currents in voltage-clamp recordings was observed in
capsaicin-sensitive cells. Capsaicin (Sigma) was dissolved in the
normal external solution containing 10% alcohol and 10% Tween 80 at a
concentration of 5 mM and then diluted in the external solution prior
to experiments. No effects were detected by application of alcohol and
Tween 80 in concentrations as high as 0.2%. SNAP (Sigma) and
nimodipine (RBI) were first dissolved in 100% DMSO at a concentration
of 500 mM, diluted in the external solution (final DMSO concentration;
0.1%). DMSO alone up to 0.2% had no effects on
Ca2+ channel currents. SNAP and nimodipine were
protected from light during the experiments.
All data are expressed as means ± SE. The data were analyzed by the Mann-Whitney U test. A level of P < 0.05 was considered to be statistically significant.
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RESULTS |
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L- and N-type Ca2+ channel currents in bladder afferent neurons
As noted in previous experiments (Yoshimura 1999;
Yoshimura and de Groat 1997a
; Yoshimura et al.
1996
), bladder afferent neurons could be divided into two
populations according to the sensitivity of their action potentials
(APs) to TTX (1 µM). Approximately 73% of bladder afferent neurons
(30 of 41 neurons) exhibited long-duration (7.9 ± 0.5 ms) APs
that were resistant to TTX in a concentration up to 6 µM; while the
remaining 11 bladder afferent neurons exhibited APs that were
reversibly blocked by 1 µM TTX (Fig. 1)
(Yoshimura 1999
; Yoshimura et al. 1996
).
In the neurons with TTX-resistant APs, the mean threshold of APs
activated by depolarizing current pulses was
20.4 ± 0.9 mV.
This type of neuron was small in size with mean diameter and cell input
capacitance of 23.6 ± 1.3 µm and 28.2 ± 1.8 pF,
respectively. In 30 bladder afferent neurons with TTX-resistant APs,
capsaicin application (1 µM) produced inward currents (1.26 ± 0.12 nA, range; 0.4-2.8 nA) at the holding potential of
60 mV in 28 neurons (93%), while the remaining 2 neurons were not sensitive to
capsaicin. In contrast, the second population of bladder afferent
neurons which exhibited TTX-sensitive APs was significantly
(P < 0.01) larger in size (diameter, 30.6 ± 1.8 µm and cell input capacitance, 38.2 ± 2.0 pF, n = 11) and had shorter (P < 0.05) duration (5.4 ± 0.5 ms) APs that were activated at significantly (P < 0.05) lower thresholds (
26.1 ± 0.9 mV) than those in
TTX-resistant neurons. Only one of these neurons was sensitive to
capsaicin (1.6 nA).
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After examining action potential properties, HVA
Ca2+ channel currents carried by
Ba2+ ions were evaluated in voltage-clamp
recordings by switching to an external solution (described in
METHODS) that suppressed Na+ and
K+ currents. In bladder afferent neurons with
TTX-resistant spikes (n = 30), L- and N-type
Ca2+ channel blockers [nimodipine (3 µM) and
-conotoxin GVIA (3 µM), respectively] inhibited 35.6 ± 3.2 and 39.2 ± 2.9% of the total HVA Ca2+
channel currents evoked by depolarizing pulses to 0 mV from the holding
potential of
60 mV (Fig. 1). In TTX-sensitive bladder afferent
neurons (n = 11), the suppression of HVA
Ca2+ channel currents by nimodipine and
-conotoxin GVIA averaged 25.8 ± 3.0 and 40.3 ± 2.9%,
respectively (Fig. 1). In addition, the peak current density of L-type
channels at 0 mV in TTX-sensitive bladder neurons was significantly
(P < 0.05) smaller (28.9 ± 4.1 pF/pA) than in
TTX-resistant bladder neurons (39.2 ± 3.8 pF/pA) while there was
no significant difference in N-type Ca2+ channel
current density in bladder afferent neurons with TTX-resistant and
-sensitive spikes (43.2 ± 3.6 and 45.1 ± 4.8 pF/pA, respectively).
Effects of SNAP on HVA Ca2+ channel currents
When an NO donor, SNAP (500 µM), was applied in the external
solution, the HVA Ca2+ channel currents evoked by
depolarizing pulses to 0 mV from the holding potential of 60 mV was
reversibly suppressed in all bladder afferent neurons tested (range;
10-40% of the peak current amplitude, n = 22 cells).
This concentration of SNAP was selected based on a previous study that
showed that concentrations ranging 100-500 µM produced significant
effects on neuronal Ca2+ channels (Chen
and Schofield 1995
; Pehl and Schmid
1997
). The suppression of Ca2+ channel
currents started within 20 s after SNAP application, reached a
plateau in 4-5 min, and recovered after washout (Fig. 2). The reduction of peak amplitude of
Ca2+ channel currents did not differ
statistically between TTX-resistant (23.4 ± 2.6%,
n = 16) and TTX-sensitive neurons (26.6 ± 3.1%, n = 6). SNAP did not change the current-voltage
relationship of HVA Ca2+ channel currents in all
22 neurons (Fig. 2C). The reproducibility of SNAP-induced
suppression of Ca2+ channel currents was tested
in six neurons, in which a second application of SNAP induced a
suppression of Ca2+ channel currents similar to
that evoked by the first application.
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Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)
To test whether SNAP inhibition of Ca2+
channel currents is associated with activation of guanylyl cyclase and
production of cGMP, the effect of SNAP on Ca2+
channel currents was tested in the presence of ODQ, an NO-selective guanylyl cyclase inhibitor (Garthwaite et al. 1995). ODQ
was applied to bladder afferent neurons prior to SNAP application.
Following a 30 min incubation with ODQ (10 µM), the peak current
density of HVA-Ca2+ channel currents in
ODQ-treated bladder afferent neurons (109.0 ± 9.5 pA/pF,
n = 7) was not different from the current density in
untreated bladder afferent neurons (112.0 ± 5.6 pA/pF; Fig. 3). In addition, in ODQ-treated bladder
afferent neurons SNAP did not suppress HVA-Ca2+
channel currents activated at 0 mV from the holding potential of
60
mV, indicating that ODQ suppressed the inhibitory effects of SNAP on
Ca2+ channel currents (Fig. 3).
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Effect of 8-bromo-cGMP
Experiments were also performed to investigate whether
8-bromo-cGMP mimicked the effect of SNAP on
HVA-Ca2+ channel currents in bladder afferent
neurons. When 1 mM 8-bromo-cGMP, a membrane permeable agent, was
applied in the external solution, Ca2+ channel
currents evoked at 0 mV from the holding potential of 60 mV were
suppressed by 28.6 ± 1.1% (n = 7 bladder
afferent neurons; Fig. 4). The
suppression of Ca2+ channel currents started
within 20 s after 8-bromo-cGMP application and reached a maximum
in 3 min.
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Channel subtypes involved in NO-mediated suppression of Ca2+ channel
SNAP-induced suppression of Ca2+ channel
currents was examined in the presence of nimodipine or -conotoxin
GVIA to block L- and N-type Ca2+ channels,
respectively, in bladder afferent neurons with TTX-resistant spikes.
Figure 5A shows that the
amount of HVA Ca2+ channel currents reduced by
SNAP (500 µM) was similar before and after inhibition of L-type
channels by nimodipine (3 µM). A comparison of the currents in the
presence of SNAP before and after nimodipine application, indicated
that only a small proportion (4.9 ± 0.7%, n = 6)
of L-type Ca2+ channel currents were suppressed
by SNAP (Fig. 5B). On the contrary, the reduction of
Ca2+ channel currents by SNAP was smaller after
an application of
-conotoxin GVIA (3 µM; Fig.
6). The suppression of N-type
Ca2+ channel currents by SNAP estimated by
subtraction of currents before and after application of
-conotoxin
averaged 54.2 ± 3.2% (n = 7 bladder afferent
neurons; Fig. 6B), indicating that SNAP preferentially
suppressed N-type Ca2+ channel currents.
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Effects of SNAP on AP characteristics
Table 1 summarizes the effects of SNAP (500 µM) on AP characteristics in bladder afferent neurons. In neurons with TTX-resistant (n = 12) and TTX-sensitive spikes (n = 5), SNAP did not influence resting membrane potential, spike threshold, spike duration and amplitude, or firing frequency during long (500 ms) depolarizing current injection.
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DISCUSSION |
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The present results indicate that application of an NO donor to bladder DRG cells can reversibly suppress HVA Ca2+ channel currents and that this effect is confined primarily to N-type Ca2+ channels. Furthermore the NO-induced suppression of Ca2+ currents appeared to be mediated by a cGMP-dependent mechanism since membrane permeable cGMP mimicked the effect of NO donors and NO-mediated suppression of Ca2+ channel currents was prevented by ODQ, an inhibitor of NO-dependent guanylyl cyclase.
It has been reported that NO exerts multiple actions on various types
of voltage-sensitive ion channels through generation of cGMP and/or
through mechanisms independent of cGMP. For example, NO donors enhance
several types of Ca2+-activated or ATP-dependent
K+ currents in smooth muscle (Koh et al.
1995) but inhibit K+ currents in neurons
from ciliary ganglia (Cetiner and Bennett 1993
). It has
also been reported that NO suppressed TTX-resistant and -sensitive
Na+ channels in baroreceptor afferent neurons
from the nodose ganglion by direct interaction of NO with the channels
(Li et al. 1998
). In other studies, NO donors increased
voltage-dependent Ca2+ currents in rat
sympathetic neurons (Chen and Schofield 1995
) but
inhibited Ca2+ currents in ciliary ganglion
neurons through cGMP-dependent pathways (Khurana and Bennett
1993
). In DRG neurons from mice and embryonic chicks, NO donors
such as sodium nitroprusside and SNAP (Ward et al. 1994
)
or a direct application of authentic NO (Kostyuk and Solovyova
1998
) inhibited voltage-dependent Ca2+
currents, although these studies were performed in unidentified cells
and the subtypes of HVA Ca2+ channels suppressed
were not investigated. Our data support the latter results and further
establish that the NO-evoked inhibition is mediated by a cGMP-dependent
signaling pathway that targets N-type Ca2+
channels in a specific population of visceral afferent neurons innervating the urinary bladder.
Voltage-sensitive Ca2+ channels are divided into
HVA and low-voltage-activated types according to their voltage
thresholds for activation. HVA channels are further classified into L,
N, P/Q, and R subtypes based on electrophysiological and
pharmacological properties (Catterall 1998;
Waterman 2000
). Bladder afferent pathways consist of
A
and C fibers that have different physiological and pharmacological
properties (Yoshimura 1999
; Yoshimura and de
Groat 1997c
). TTX resistance and capsaicin sensitivity occur
primarily in small-sized C-fiber afferent neurons; whereas
TTX-sensitive Na+ channels and capsaicin
resistance are properties of the larger A
afferent cells
(Yoshimura 1999
; Yoshimura and de Groat
1999
). The present study revealed that N- and L-type channels
account for approximately 70% of the total HVA
Ca2+ current in bladder afferent neurons and that
the density of L-type channel currents in TTX-resistant bladder
afferent neurons is larger than in TTX-sensitive neurons. Although P/Q
channel subtypes sensitive to agatoxin IVA were not investigated, it
seems reasonable to conclude that N and L channels are major subtypes
of HVA Ca2+ channels in both types of bladder
afferent neurons, expression of L-type Ca2+
channels is greater in C-fiber bladder afferent neurons than in
A
-fiber neurons, and the proportion of N-type channels is similar in
the two types of neurons. This is in line with previous findings in
unidentified DRG neurons in which L-type Ca2+
currents were more prominent in small-diameter DRG neurons than in
medium/large diameter neurons (Scroggs and Fox 1992
).
While it has been documented that low-voltage-activated T-type channels
are important in controlling cell excitability, HVA Ca2+ channels are known to be involved in
neurotransmitter release in nerve terminals (Catterall
1998; Waterman 2000
). It has been postulated
that Ca2+ entry via N-type channels mediates
release from small vesicles containing classical transmitters such as
norepinephrine while Ca2+ influx through L-type
channels may favor release from large dense core vesicles containing
neuropeptides (Hirning et al. 1988
). Since C-fiber
afferent nerves likely contain neuropeptides such as substance P or
CGRP, the prominence of L-type currents in C-fiber bladder afferent
neurons with TTX-resistant spikes very likely contributes to peptide
release from the nerve terminals. However, recent studies have
demonstrated that N-type Ca2+ channels in sensory
nerves including bladder afferents are also important for the release
of neuropeptides induced by electrical stimulation or application of
bradykinin, whereas K+-induced release was
dependent on L-type channels (Maggi et al. 1989
, 1990
;
Waterman 2000
). Therefore NO-mediated suppression of
N-type Ca2+ channels raises the possibility that
NO might modulate the release of various transmitters from afferent
nerve terminals in the bladder. If this occurs, it seems likely that
both A
- and C-fiber bladder afferent nerves would be affected
because SNAP suppressed HVA Ca2+ currents in both
TTX-resistant and -sensitive neurons.
An action of NO on afferent nerve terminals is consistent with the
results of our recent in vivo study, which revealed that NO donors
administered into the bladder suppressed bladder hyperactivity elicited
by CYP-induced cystitis in the rat (Ozawa et al. 1999). Because the bladder smooth muscle in the rat is relatively insensitive to NO (Andersson 1993
), it was proposed that the
depressant effect of the NO donor was due to an inhibitory action on
afferent nerves that were sensitized by the cystitis. However, in the
present experiments, SNAP did not alter the resting membrane potential or the threshold for initiation of AP, suggesting that NO may not
directly affect firing properties of bladder afferent neurons but may
act indirectly by inhibiting N-type Ca2+ channels
and in turn the release of afferent neurotransmitters that amplify the
inflammatory conditions in the bladder (Burnstock 1999
;
Maggi et al. 1989
).
NO might be released from multiple sites in the bladder. The
NO-synthesizing enzyme, nitric oxide synthase (NOS), and NADPH diaphorase, a chemical marker for NOS, are localized in afferent and
efferent neurons innervating the urinary tract (Vizzard et al.
1993). At least three types of NOS have been identified.
Neuronal (nNOS) and endothelial NOS (eNOS), which are constitutive and Ca2+/calmodulin-dependent, release NO for short
periods in response to stimulation. The other enzyme is inducible
(iNOS), is Ca2+-independent, and, once expressed,
generates large amounts of NO for long periods (Moncada et al.
1991
). NOS activity in the normal bladder is mainly due to the
constitutive NOS isoforms (more than 95%). eNOS has been detected in
urothelial cells. NO can also be released from these cells by various
chemical stimuli (e.g., norepinephrine, acetylcholine, and capsaicin)
(Birder et al. 1998
). High basal release of NO mediated
by iNOS has been detected in bladder strips from rats with CYP-induced
cystitis (Birder et al. 1997
). Previous studies in rats
have also demonstrated that chronic bladder inflammation upregulates
the expression of NOS in bladder afferent neurons (Vizzard et
al. 1996
) and that suppression of NO release in the spinal cord
by a NOS inhibitor suppressed cystitis-induced bladder hyperactivity
(Kakizaki and de Groat 1996
; Rice 1995
).
Thus it seems likely that increased generation of NO in afferent nerve
terminals in the spinal cord may be involved in enhancing reflex
activity and/or inflammatory responses of the bladder.
However, NO is also proposed as a mediator that suppresses tissue
inflammation or injury in visceral organs such as intestine and
bladder. For example, experimental studies revealed that NO donors can
reduce the severity of gastric and intestinal injury induced by ethanol
application or ischemia (Muscará and Wallace 1999). Recent clinical studies also demonstrated that
production of NO in the bladder is decreased in patients with
interstitial cystitis, a painful bladder syndrome of unknown etiology
(Smith et al. 1996
), and that orally administered
L-arginine, an NO precursor, was effective in reducing
irritable bladder symptoms in these patients (Smith et al.
1997
). Furthermore, as mentioned in the preceding text, NO
donors injected into the bladder inhibited bladder hyperactivity
elicited by CYP-induced inflammation, probably due to suppression of
bladder afferent pathways (Ozawa et al. 1999
). Thus it
is reasonable to assume that NO has a dual effect, possibly dependent
on concentration or different sites of action (i.e., central or
peripheral), to suppress or amplify nociceptive mechanisms
(Colasanti and Suzuki 2000
). The NO-mediated protective mechanism might involve suppression of N-type
Ca2+ channels and subsequent neuropeptide release
from bladder sensory nerves as suggested from the present study.
Therefore manipulation of NO levels or NO-cGMP signaling mechanisms
could provide a new therapeutic modality for treating hyperactivity
and/or painful sensations in visceral organs such as the urinary bladder.
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ACKNOWLEDGMENTS |
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This study was aided by Paralyzed Veterans of America Spinal Cord Research Foundation Grant 1861-01/02 and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-49430 and DK-57267.
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FOOTNOTES |
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Address for reprint requests: N. Yoshimura, Dept. of Pharmacology, University of Pittsburgh School of Medicine, W1353 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: nyos{at}pitt.edu).
Received 11 January 2001; accepted in final form 27 March 2001.
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REFERENCES |
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