Departments of 1 Medical Research, 2 Urology, 3 Medicine, and 4 Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 10022; and 5 Department of Physiology, Chung Shan Medical and Dental College, Taichung, Taiwan
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ABSTRACT |
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We explored whether substance P (SP) via neurokinin (NK) receptor facilitates bladder afferent signaling and reactive oxygen species (ROS) formation in bladder in association with neurogenic inflammation. We evaluated ROS activity and cystometrograms as well as pelvic nervous activity in anesthetized rat bladder with SP stimulation. Our results showed that endogenous SP via NK1, not NK2, receptor mediated a micturition reflex. An increase in SP by electrical stimulation of the pelvic nerve or an increase in exogenous SP by intra-arterial or intrathecal administration can facilitate myogenic and neurogenic bladder contractions. Furthermore, exaggerated SP release increased ROS in the bladder and whole blood via increased mast cell degranulation, intercellular adhesion molecule expression, and leukocyte adhesion, a primary source of ROS in the inflamed bladder. Treatment with NK1-receptor antagonists or ROS scavengers reduced bladder intercellular adhesion molecule expression and ROS and ameliorated the hyperactive bladder response. Our study indicates that the mechanism by which SP participates in the neurogenic bladder may be complicated by its proinflammatory activity and its ability to stimulate ROS generation.
reactive oxygen species; micturition reflex; neurokinin receptor
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INTRODUCTION |
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THE TACHYKININS SUBSTANCE P (SP) and neurokinins (NK) A and B belong to a family of neuropeptides that are widely distributed in the mammalian central and peripheral afferent nervous systems and produce their biological actions by activating three distinct receptor types, NK1, NK2, and NK3 (25). These afferents commonly innervate smooth muscle, submucosal layers, and blood vessels of visceral organs (4, 21, 34). On release from sensory afferents, SP and NKA, via NK1 and NK2 receptors, act on smooth muscle or blood vessels to regulate visceral motility and blood flow (21, 25). Recent evidence suggests that endogenous tachykinins may play a role in visceral inflammation, hyperreflexia, and hyperalgesia (4, 20). For example, in the rat urinary bladder, SP may be more relevant than NKA for the mediation of plasma protein extravasation and inflammatory response (19), whereas NKA is an important mediator of smooth muscle contraction (21). In addition, tachykinins may lead to expression of adhesion molecules by endothelial cells, chemotaxis and activation of immune cells, mucus secretion, and water absorption/secretion in the lungs, gastrointestinal tract, and genitourinary tract (1, 19, 32).
The mammalian bladder is richly innervated by capsaicin-sensitive, SP-containing afferent fibers (22, 37). Afferents signaling mechanical and chemical environments in the bladder evoke sacrolumbar reflexes in lumbar sympathetic neurons to increase bladder capacity or elicit parasympathetic bladder efferent excitation to trigger a normal micturition reflex (8, 9, 11). However, the bladder capacity and/or the micturition reflex may be altered in a number of pathophysiological conditions, such as interstitial cystitis, cyclophosphamide-induced cystitis, and irritant-induced hypersensitivity (14). The mechanisms responsible for bladder hyperactivity may vary from condition to condition and are likely complicated. Bladder biopsies in some patients diagnosed with interstitial cystitis have shown increased density of SP-containing fibers and NK1 receptors (15, 26, 29). Furthermore, administration of SP is known to cause bladder inflammation (1, 21, 39) and generation of reactive oxygen species (ROS) by inflammatory cells (3, 40). Thus SP may have a direct or an indirect role in neurogenic inflammation and regulation of bladder motility in various clinical conditions. Nonetheless, whether ROS generated by SP-reacted inflammatory cells contribute to bladder hyperactivity remains to be determined.
Our objective in the present study was to clarify the contribution and the possible mechanism of SP in bladder hyperreflexia. We showed that bladder hyperactivity caused by pelvic nerve stimulation is associated with an increased SP level in the bladder. We also used NK-receptor antagonists and free radical scavengers to test the role of SP- and ROS-induced hyperactivity, respectively. Our results clearly showed that SP via NK1-receptor activation enhances the micturition reflex and ROS release from the inflammatory cells and, consequently, leads to a hyperactive bladder.
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MATERIALS AND METHODS |
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Drugs.
The drugs/chemicals used in this study are listed in Table
1. The drugs were prepared and stored at
70°C, and subsequent dilutions of the drugs were made in saline on
the day of the experiments.
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Surgery. Adult female Wistar rats weighing 220-240 g were anesthetized with urethane (1.2 g/kg sc), which is well known to anesthetize the animals yet permit full reflex bladder contractions (8, 9). Maintenance of deep anesthesia was determined by the persistence of miotic pupils as judged from frequent inspection and by the lack of heart rate and arterial blood pressure (ABP) fluctuations in the absence of visceral stimuli (8). An experiment was terminated when the baseline mean ABP was <90 mmHg. Animal care and experimental protocol were in accordance with the guidelines of the National Science Council of the Republic of China (1997). All efforts were made to minimize animal suffering and the number of animals used throughout the experiment. At the end of each experiment, the animals were killed by an intravenous potassium chloride injection.
PE-50 catheters were placed in the left femoral artery for measurement of ABP and in the left femoral vein for administration of anesthetics. ABP was recorded on a polygraph (model RS3400, Gould) with a transducer (model P23 1D, Gould-Statham, Quincy, MA). A length of stretched PE-10 tubing inserted just above the bifurcation of the aorta from the right femoral artery was used for injection of various drugs. Body temperature was kept at 36.5-37°C by an infrared light and was monitored with a rectal thermometer.Experimental models and protocols. We used a transcystometer (via cannulation of the bladder dome) mimicking normal micturition of the bladder to evaluate the role of SP in the bladder micturition reflex and bladder hyperactivity (8, 18). Briefly, the urinary bladder was exposed through a midline incision of the abdomen, and urine was emptied by application of light pressure. A PE-50 T tube was inserted through the apex of the bladder dome. The bladders were filled by continuous infusion of 0.9% saline (0.15 ml/min) at room temperature and allowed to drain/micturate repeatedly via the urethra.
The change in bladder pelvic afferent nerve activity (PANA), bladder pelvic efferent nerve activity (PENA), ABP, and various parameters measured by the cystometrogram can be determined before and after intra-arterial or intrathecal administration of various chemicals. All chemicals were injected through the intra-arterial catheter in a volume of 1 ml/kg (0.20-0.25 ml) and were followed by 0.1 ml of heparinized saline. For intrathecal administration, a catheter (Portex, Hythe, Kent, UK) was inserted through the atlantooccipital membrane for 8.5 cm, such that the tip of the catheter was placed just above the lumbosacral enlargement, as described previously (18). Chemicals were given intrathecally in a volume of 20 µl and were followed by 30 µl of saline. In the first part of study, the animals were divided into three groups: group 1 was used to test the role of endogenous SP in the bladder micturition reflex by administration of NK-receptor antagonists; groups 2 and 3 were used to test the effect of exogenous SP and SP + NK-receptor antagonists on bladder hyperactivity. SP was given at 0.1-10 µg ia and 5 µg it. Plasma SP in the iliac vein was 35-1,200 ng/ml at 10 min after intra-arterial injection. To verify the SP activity via the NK1 receptor, we injected SP (group 2), SP + the NK1-receptor antagonist CP-96345 (group 3a), or the NK2-receptor antagonist SR-48968 (group 3b). The NK-receptor antagonists were given at 250-500 µg ia and 5-250 µg it, doses that were lower than those used in previous studies (Cystometrogram for measurement of bladder response.
The bladder catheter was connected via a T tube to a pressure
transducer (model P23 1D, Gould-Statham), and the intravesical pressure
(IVP) was recorded continuously on a polygraph (model RS3400, Gould,
Cleveland, OH). The following parameters of bladder responsiveness were
also measured: threshold volume (infused volume at the point preceding
a micturition reflex), number of active contractions (>15 mmHg),
micturition volume (volume of expelled urine collected in a preweighed
tube), residual volume (amount of fluid remaining after a bladder
contraction; infused volume = infusion rate micturition
volume), bladder capacity (residual volume + micturition volume),
intercontraction interval (time lag between 2 micturition cycles), and
basal pressures.
Recording of PANA and PENA. Multifiber PANA and PENA were measured simultaneously in eight rats. The two left pelvic nerve branches attached to the urinary bladder surface were isolated and simultaneously recorded by placement of the intact nerve fibers in parallel with two pairs of thin, bipolar stainless steel electrodes (8). The electrical signals were amplified 20,000-fold, filtered (high-frequency cutoff at 3,000 Hz and low-frequency cutoff at 30 Hz) with an alternating-current preamplifier (model P511, Grass, Valley View, OH), continuously recorded on magnetic tape, and displayed on an oscilloscope (model 1604, Gould). The amplified signals (spikes) were transformed by a window discriminator (model 121, World Precision Instruments, Sarasota, FL) and analyzed with an impulse counter (Gould integrator amplifier 13-4615-70) that was set to count the total number of spikes per second (8, 9). The background activity, which could be caused by the nerve contact with electrodes, nerve damage during handling, and the equipment itself, was excluded from the window discriminator by adjustment of the threshold voltage (9).
Nerve fiber with PANA was confirmed by its ability to show increased activity in response to small increments in IVP by saline infusion via T tube. Nerve fiber with PENA had minimal activity until a threshold volume/pressure in the bladder produced a bursting discharge causing a micturition contraction (Fig. 1) (8, 9, 36).
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Electrical stimulation of the pelvic nerve. The next study was intended to determine whether nerve activity causing bladder hyperactivity was accompanied by release and/or elevation of SP in bladder. An electric current of square-wave pulses with pulse duration of 0.05 ms was applied from a stimulator (model S88, Grass, Quincy, MA) through a stimulus isolation unit (model SIU5B, Grass) and a constant-current unit (model CCU1A, Grass).
Six rats were given a solution of atropine (1 mg · mlMeasurement of SP.
Plasma levels of SP in the iliac vein were measured as described
previously (5). The study was intended to determine
whether pelvic nerve activity results in elevation and release of SP in the bladder. Briefly, supernatant from plasma samples was diluted with
the same volume of buffer A (RIK-BA-1, Peninsula
Laboratory). Then each sample was passed slowly through a C18 Sep-Pak
column (RIK-SEPCOL-1, Peninsula Laboratory). The column was washed with 9 ml of buffer A and eluted with 3 ml of buffer B
(RIK-BB-1, Peninsula Laboratory). The eluted samples were dried by
vacuum centrifugation and stored at 70°C for later analysis. An SP
enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) was used to
detect the SP level. Each sample was dissolved in 1% HCl, diluted to a
suitable concentration with enzyme immunoassay buffer, and assayed in
duplicate. The SP, which was linked to acetylcholinesterase as a
tracer, and rabbit SP antiserum were added to the sample and incubated in the assay plate at 4°C for 18 h. Then, the wells were rinsed five times with washing buffer. Ellman's reagent was added for development of the plates in each well. After development, the plates
were read at 410 nm, and SP levels were calculated.
Detection of ROS production in urinary bladder after exogenous SP administration. SP is capable of causing intracellular ROS generation and release by inflammatory cells and, perhaps, other types of cells (3, 40). Thus we want to examine whether ROS generated after exogenous SP administration contributes to bladder hyperactivity. The ROS generation in response to SP stimulation was measured in whole blood (obtained from the femoral artery) and bladder by a chemiluminescence (CL) detection method as described previously (7). In addition, the possible cellular source of ROS in the urinary bladder was examined by the fluorescence emitted after dichlorofluorescin (DCFH) diacetate infusion (38).
A change in the bladder surface during the filling and micturition states of the transcystometric model may affect the measurement of ROS. Thus an isovolumetric model allowing minimal bladder surface change was adopted for measurement of ROS generation from the bladder in vivo. Eighteen rats were used and grouped (see below) in this part of the experiment. To establish an isovolumetric condition, we inserted one PE-50 tube into the bladder through the urethra and tied it in place with a ligature around the urethral orifice (23). The catheter was connected to a separate pressure transducer and an infusion pump via a T tube connector. Transurethral filling (0.15 ml/min) of 0.9% saline into the urinary bladder via the urethral catheter was done until rhythmic bladder contractions occurred. The infusion was stopped, and the bladder was maintained under constant-volume conditions by ligation of the ureter bilaterally. The method for detection of ROS from the organ surface after 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-(1,2-a)-pyrazin-3-one hydrochloride administration (0.2 mg · mlCellular origin of ROS in the bladder after SP administration. We conducted DCFH diacetate tissue staining to determine the cellular origin of ROS in the bladder after SP administration. DCFH diacetate is a stable nonfluorescent compound that can diffuse into cells, is hydrolyzed to DCFH, and is thereby trapped within the cells (38). DCFH is oxidized by ROS to yield dichlorofluorescein (DCF), a fluorescent molecule (38).
At 10 min after SP (10 µg ia) administration, the bladder was filled transurethrally with 5 µM DCFH diacetate (Sigma, St. Louis, MO) and 1 µM propidium iodide (PI; Sigma) in a volume of 0.5 ml of saline for an additional 30 min and then washed out with saline. The bladder was removed, cut, and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA). Frozen sections (10 µm thick) were obtained, and cellular fluorescence intensity in the tissue sections was examined by fluorescence microscopy (model DMRD, Leica Microsystems Wetzlar, Wetzlar, Germany). In addition, a portion of the urinary bladder was cut and fixed in 10% neutral buffered formalin solution, dehydrated in graded ethanol, and embedded in paraffin. Sections (5 µm) of bladders were stained with hematoxylin and eosin and Giemsa and evaluated for the extent of inflammatory cell accumulation and number of mast cells (31).Effects of SP-induced ROS formation on bladder hyperactivity.
We detected formation of ROS in the bladder of SP-treated rats. Next,
we performed studies to determine the role of ROS in bladder
hyperactivity. Before SP stimulation, baseline ABP, IVP, and ROS
amounts were recorded for 30 min as a control value. After 10 min of SP
stimulation, the rats (n = 18) were divided into groups
and treated with saline (n = 2), CP-96345 (500 µg,
n = 4), SR-48968 (500 µg, n = 2),
superoxide dismutase (SOD, 500 U, n = 4),
FC4S (250 µg, n = 3), or C3
(250 µg, n = 3). The three antioxidants (SOD and the
fullerenes FC4S and C3) were used to test
whether the SP-induced bladder hyperactivity can be ameliorated by free
radical scavengers. The ABP and cystometrogram were monitored simultaneously for 60 min. After measurement, the bladder tissues and
leukocytes isolated from 4 ml of whole blood were collected and stored
at 70°C for immunoblotting analysis.
Immunoblot analysis for ICAM and -actin.
The immunoblotting method was described previously (7). We
measured the amounts of ICAM and
-actin in bladder tissues and
leukocytes of SP-treated rats. For protein analysis, bladder samples
and leukocytes were homogenized with a prechilled mortar and pestle in
extraction buffer, which consisted of 10 mM Tris · HCl (pH
7.6), 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40, 0.5%
deoxycholate, 2%
-mercaptoethanol, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin. The mixtures were homogenized completely by
vortexing and kept at 4°C for 30 min. The homogenate was centrifuged at 12,000 g for 12 min at 4°C, the supernatant was
collected, and protein concentration was determined by Bio-Rad protein
assay (Bio-Rad Laboratories, Hercules, CA). Antibodies raised against ICAM (catalog no. AF583, R & D Systems, Minneapolis, MN) and
-actin (catalog no. A5316, clone AC-74, Sigma) were used. Both of these antibodies cross-react with respective rat antigens.
Data acquisition and statistical analysis. All nervous activity (PANA and PENA) was expressed as the number of spikes per second. We analyzed the PANA and the frequency of bursting PENA before and after drug treatment. Values are means ± SE. Data were subjected to analysis of variance, followed by Duncan's multiple-range test for assessment of the differences among groups. Student's paired t-test was used to detect differences between control and drug treatment. P < 0.05 was considered to indicate statistical significance.
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RESULTS |
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The PANA and PENA, IVP, and ABP in rats with normal micturition reflex were recorded simultaneously. The IVP was gradually increased on accumulation of saline in the urinary bladder, causing activation of PANA. The frequency of PANA over time was progressively increased by the increase in bladder filling volume. When a threshold volume (~0.42-0.62 ml, mean 0.5 ml) was reached to evoke a micturition reflex, PANA quickly reached its peak (i.e., enhanced PANA), and then a bursting efferent discharge (PENA) that lasted for 7-15 s was detected (Fig. 1). The bursting PENA can simultaneously trigger an active neural reflex-mediated bladder contraction, as shown by a quick rise in IVP. ABP was slightly and transiently elevated (for ~15-35 s) in accordance with the abrupt rise in IVP and the neurogenic bladder contraction (Fig. 1).
Endogenous SP via NK1 receptor participates in a normal
micturition reflex.
Intra-arterial administration of CP-96345 (250 µg) significantly
decreased the frequency of PANA over time, the frequency of bursting
PENA, and active contractions (P < 0.05) and increased bladder capacity (Fig. 2, Table
2; P < 0.05). This result suggested that intra-arterial CP-96345 exerts a
partial inhibition on afferent neurotransmission of the micturition
reflex to increase the bladder capacity. A more significant effect of
CP-96345 was detected when it was administered intrathecally. The mean
frequency of PANA over time increased after intrathecal CP-96345
administration, while the peak frequency of PENA dropped to zero,
indicating that lumbosacral spinal NK1-receptor blockade
completely inhibited the afferent and efferent neural transmission of
the micturition reflex. Inhibition in micturition parameters is
displayed as a function of dose (Fig. 3,
Table 2). The abrupt and dose-dependent hypotensive response to
intrathecal administration of the NK1 antagonist indicates
CP-96345 leakage into the systemic circulation. In contrast,
intra-arterial or intrathecal administration of SR-48968 had no
influence on the micturition reflex (Table 2), suggesting that SP via
mainly the NK1, not the NK2, receptor mediates
the afferent micturition pathway in the lumbosacral spinal cord (Fig. 2, right, and Fig. 3C).
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Pelvic nerve stimulation enhances bladder hyperactivity and SP
release.
In rats, an increase in frequency (1-10 Hz) of pelvic nerve
stimulation significantly enhanced the duration of bladder contraction, decreased intercontraction intervals (1.6 ± 0.4, 0.6 ± 0.2, 0.4 ± 0.1, and 0.3 ± 0.1 min at 0, 1, 5, and 10 Hz,
respectively), and, at the same time, increased the release of SP
to the bladder outflow (59 ± 8, 94 ± 14, 190 ± 30, and 254 ± 36 ng/ml at 0, 1, 5, and 10 Hz, respectively; Fig.
4). The data clearly showed that bladder
hyperactivity caused by pelvic nerve stimulation is associated with a
dose-dependent increase in SP in the bladder. Furthermore, we suggest
that, in addition to SP, NKA and other substances are released from
sensory pelvic afferents during electrical stimulation and,
consequently, lead to a prolonged bladder contraction.
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Exogenous SP facilitates the micturition reflex in bladder
hyperreflexia.
At residual volume immediately after micturition, no significant PANA
and PENA were observed, and the IVP remained at the basal level when
saline infusion was stopped (Fig.
5A). At ~5-10 s after
intra-arterial administration of SP (0.1-5 µg), the bladder revealed an elevated IVP and dose-dependently enhanced PANA and PENA
(Fig. 5B, fast trace).
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Increased ROS formation from whole blood and bladder surface after
exogenous SP administration.
The SP-treated whole blood samples showed a dose-dependent increase in
ROS formation: 78 ± 20, 336 ± 67, 560 ± 105, 1,035 ± 213, 1,201 ± 196, and 1,108 ± 230 counts/10 s
at 0, 0.1, 1, 10, 50, and 100 µg of SP, respectively (Fig.
7A). The source of whole blood
ROS was leukocytes, not erythrocytes and plasma (Fig. 7C).
Coincubation with the NK1-receptor antagonist CP-96345 (50 µg) prevented SP-induced ROS generation from whole blood, which was
reduced from 1,065 ± 210 to 97 ± 18 counts/10 s in response to treatment with 50 µg of SP (Fig. 7B). In contrast,
SR-48968 had no significant inhibitory effect on SP-induced ROS
generation: 1,065 ± 210 vs. 899 ± 177 counts/10
s (P > 0.05). Coincubation with the free radical
scavengers SOD, C3, and FC4S also significantly reduced the SP-induced ROS generation: 220 ± 51, 430 ± 89, and 190 ± 56 counts/10 s, respectively (P < 0.05). The magnitude of the reduction exhibited by free radical
scavengers was slightly lower than that exhibited by CP-96345,
suggesting that directly blocking upstream NK1-receptor
activation by a long-lasting effect of the nonpeptide CP-96345 is more
efficient than scavenging downstream ROS activity by free radical
scavengers.
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Inhibition of bladder hyperactivity by free radical scavengers.
In the isovolumetric condition without SP stimulation, the frequency of
bladder contractions was 3.4 ± 0.5 active contractions/10 min,
and the basal level of bladder ROS was maintained at ~140 ± 25 counts/10 s. Intra-arterial SP stimulation significantly reduced ABP
(from 124 ± 5 to 78 ± 6 mmHg, P < 0.05)
and increased the frequency of active contractions (7.3 ± 1.7 active contractions/10 min, P < 0.05) and the bladder
ROS generation (Fig. 8A; from
140 ± 25 counts/10 s before SP stimulation to 2,560 ± 345 counts/10 s after 10 min of SP, P < 0.05). At 10 min
after SP stimulation, intra-arterial saline had no effect on the
SP-induced response. The response of the hyperactive bladder and the
increase in ROS generation could be maintained for >30 min.
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Cellular origin of ROS in SP-induced bladder inflammation.
Sections from the bladder mucosa and smooth muscle obtained
40-45 min after SP administration revealed infiltration of
leukocytes and the presence of several degranulated mast cells (Fig.
9). Sections of urinary bladders from
control animals exhibited no signs of inflammation at the end of the
experiments.
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Effect of NK-receptor antagonist or SOD on ICAM expression in
SP-treated bladder and leukocytes.
Expression of ICAM and -actin in the bladder and leukocytes after SP
treatment was assessed by immunoblotting with antibodies against ICAM
and
-actin (Fig. 10). ICAM
expression was detected in control bladder and leukocytes. ICAM
expression in bladder tissues was dose dependently increased by SP
stimulation: 1.0 ± 0-, 2.0 ± 0.3-, and 3.5 ± 0.5-fold
for control, 1 µg of SP, and 10 µg of SP, respectively. However,
the enhanced ICAM expression was significantly inhibited by CP-96345
(from 3.5 ± 0.5- to 1.3 ± 0.3-fold) or SOD (from 3.5 ± 0.5- to 1.9 ± 0.3-fold). SR-48968 had no effect on enhanced
ICAM expression (3.5 ± 0.5- vs. 3.1 ± 0.6-fold). ICAM
expression in leukocytes was unaffected by SP stimulation.
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DISCUSSION |
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The present study demonstrates for the first time the involvement
of ROS generation in SP-mediated bladder hyperreflexia. SP is a sensory
neuropeptide present in small myelinated A-fibers and in
small-diameter unmyelinated C fibers (22). Once released from bladder afferent nerves and the sacral spinal cord, SP is involved
in the mechanoreceptor-mediated micturition reflex. In rats, systemic
administration of capsaicin for depletion of SP resulted in urine
retention or an increased volume/pressure threshold for micturition,
implicating an excitatory role of SP in the afferent micturition
pathway (6, 22). We demonstrated that intra-arterial administration of exogenous SP could initiate the micturition reflex
response in bladders containing a subthreshold amount (residual volume)
of fluid. The SP-mediated micturition reflex or bladder hyperactivity
is mainly through NK1 receptors. We further showed that
exogenous or excessive SP stimulation also resulted in increased ROS
generation. NK1-receptor blockade or free radical
scavengers could inhibit micturition pathway facilitation and
SP-mediated ROS generation and, subsequently, ameliorate SP-induced
bladder hyperactivity.
In our study, the cystometrogram and electrophysiological recording techniques in urethane-anesthetized rats are valuable tools for analyzing the neural control of the urinary bladder (6, 9, 36). Efficient bladder voiding is triggered primarily by the cumulative afferent activities from the bladder mechanoreceptors during bladder distension, which subsequently elicits parasympathetic bladder efferent excitation to evoke a micturition reflex when a threshold volume/pressure is reached (8 9). A bladder hyperreflexia or hyperactivity indicates initiation of a micturition reflex response, even in bladders containing a subthreshold amount (residual volume) of fluid. A normal bladder contraction is coordinated by efferent purinergic, cholinergic, muscarinic, and nicotinic (somatic) elements (9). In addition, several neuropeptides (e.g., SP and NKA) have been demonstrated in the rat urinary bladder, yet their roles in normal micturition and bladder hyperactivity remain to be further elucidated (21).
Stimulation by exogenous SP or NK-receptor agonists has been shown to
induce bladder hyperactivity (18, 21, 22). Maggi (22) demonstrated facilitation of reflex micturition by
intravesical administration of [-Ala8]-NKA (an
NK2-receptor agonist), suggesting a peripheral site of
action via smooth muscle contraction in the bladder. SP can also be a
centrally excitatory neurotransmitter producing a slow excitatory
postsynaptic potential in the neurons of the spinal cord and mediating
a dorsal root C fiber reflex to the dorsal horn neurons, leading to
bladder hyperreflexia (Fig. 3, Table 2). Thus SP exerts effects on the
peripheral bladder and the central spinal cord to facilitate the
micturition pathway and, consequently, lead to bladder hyperactivity.
The SP-mediated hyperactivity can be inhibited by intra-arterial or
intrathecal administration of CP-96345 (an NK1-receptor
antagonist), but not SR-48968 (an NK2-receptor antagonist).
It is also known that a peripheral release of tachykinins determines a
set of responses (loosely defined as neurogenic inflammation) that
includes vasodilatation, plasma protein extravasation, smooth muscle
contraction, stimulation of afferents, and inflammation (19). To further complicate matters, sensory neurons and
immune cells can express and release tachykinins, which may also
contribute to neurogenic inflammation in the bladder. SP (0.3-1
µM) is able to induce, in a dose-dependent manner, secretion of
various cytokines (e.g., interleukins-1 and -6 and tumor necrosis
factor-) from cultured lymphocyte-enriched mononuclear cells
isolated from human peripheral blood (10). In addition, SP
has been shown to cause a proinflammatory change in tissues, such as
degranulation of mast cells and leukocyte adhesion to the venular
endothelium (39), by a mechanism of SP-enhanced ICAM
expression in the bladder tissue (Fig. 10). The possible involvement of
SP and its NK1 receptor in pathophysiological changes of
bladder inflammation has been underscored by a recent study showing a
dramatic reduction in antigen-induced cystitis in
NK1-receptor-deficient mice (31).
As a consequence of SP-immune and -inflammatory cell interaction, a variety of substances, such as histamine, cytokines, and ROS, are released (3, 10, 32, 39). In whole blood and leukocytes incubated with SP, ROS activity was displayed in a dose-dependent manner and SP-induced ROS release and ICAM expression are inhibited by CP-96345, but not SR-48968, confirming a mediating role of the NK1 receptor in triggering ROS generation in leukocytes. We further showed that SP-induced ROS generation in the bladder as well as bladder hyperreflexia can be partly ameliorated by CP-96345 or, noticeably, by free radical scavengers. It is not totally unexpected that SP-induced ROS formation may contribute to the myogenic and neural hyperactivity. ROS are known to be involved in changes in muscle tone, vascular smooth muscle strip contraction (2, 30), and increased neural activity/conduction velocity in vitro by mechanisms such as alterations in membrane conductance, calcium homeostasis, calcium-dependent processes, and eicosanoid and nitric oxide metabolism (2, 16, 42).
In summary, our studies provided direct evidence that SP participates in the micturition reflex response in bladders by activating NK receptors to facilitate the afferent pathway. Increased SP stimulation may enhance the afferent discharge (PANA) to the central nervous system to result in a hyperactive bladder. On the other hand, increased nerve activity by other means may result in increased release of SP, further complicating bladder hyperactivity. Our study indicates that the mechanism by which SP participates in the neurogenic bladder may be complicated by its proinflammatory activity and its ability to stimulate ROS generation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Taiwan University Hospital Grants NTUH89A014, 89A023-10, and 89A023-11, National Science Council of the Republic of China Grants NSC90-2320-B-002-035, NSC90-2320-B002-152, NSC90-2314-B-002-446, and NSC89-2314-B-002-350, and the Mrs. Hsiu-Chin Lee Kidney Research Fund (to C.-T. Chien).
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FOOTNOTES |
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* C.-T. Chien and H.-J. Yu contributed equally to this work.
Address for reprint requests and other correspondence: C.-T. Chien, Dept. of Medical Research, National Taiwan University Hospital and National Taiwan University College of Medicine, 1-1, Jen-Ai Rd., Taipei, Taiwan, ROC (E-mail: ctchien{at}ha.mc.ntu.edu.tw).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00187.2002
Received 14 May 2002; accepted in final form 3 December 2002.
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