Substance P via NK1 receptor facilitateshyperactive bladder afferent signaling - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:34 |只看该作者 |倒序浏览 |打印

作者：Chiang-TingChien, Hong-JengYu, Tser-BinLin, Ming-KuenLai,  Su-MingHsu作者单位：Departments of Medical Research, Urology, Medicine, and Pathology, National TaiwanUniversity Hospital and National Taiwan University College of Medicine,Taipei 10022; and Department of Physiology, Chung ShanMedical and Dental College, Taichung, Taiwan 5 r- B2 ~% f$ Y) _) [! l: N0 B$ w; B
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      【摘要】2 E. r" A$ n* ^* M
   We explored whether substance P (SP) vianeurokinin (NK) receptor facilitates bladder afferent signaling andreactive oxygen species (ROS) formation in bladder in association withneurogenic inflammation. We evaluated ROS activity and cystometrogramsas well as pelvic nervous activity in anesthetized rat bladder with SPstimulation. Our results showed that endogenous SP via NK 1, not NK 2, receptor mediated a micturition reflex. Anincrease in SP by electrical stimulation of the pelvic nerve or anincrease in exogenous SP by intra-arterial or intrathecaladministration can facilitate myogenic and neurogenic bladdercontractions. Furthermore, exaggerated SP release increased ROS in thebladder and whole blood via increased mast cell degranulation,intercellular adhesion molecule expression, and leukocyte adhesion, aprimary source of ROS in the inflamed bladder. Treatment withNK 1 -receptor antagonists or ROS scavengers reduced bladderintercellular adhesion molecule expression and ROS and ameliorated thehyperactive bladder response. Our study indicates that the mechanism bywhich SP participates in the neurogenic bladder may be complicated byits proinflammatory activity and its ability to stimulate ROS generation. + }1 `! B3 F& ]# n/ j% E* {& g
      【关键词】 reactive oxygen species micturition reflex neurokininreceptor
   INTRODUCTION1 o: A( l& ]; ]. V7 [# k

THE TACHYKININS SUBSTANCE P (SP) and neurokinins (NK) A and B belongto a family of neuropeptides that are widely distributed in themammalian central and peripheral afferent nervous systems and producetheir biological actions by activating three distinct receptor types,NK 1, NK 2, and NK 3 ( 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, viaNK 1 and NK 2 receptors, act on smooth muscle orblood vessels to regulate visceral motility and blood flow ( 21, 25 ). Recent evidence suggests that endogenous tachykinins mayplay 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 NKAis an important mediator of smooth muscle contraction( 21 ). In addition, tachykinins may lead to expression ofadhesion molecules by endothelial cells, chemotaxis and activation ofimmune cells, mucus secretion, and water absorption/secretion in thelungs, gastrointestinal tract, and genitourinary tract ( 1, 19, 32 ).
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The mammalian bladder is richly innervated by capsaicin-sensitive,SP-containing afferent fibers ( 22, 37 ). Afferentssignaling mechanical and chemical environments in the bladder evokesacrolumbar reflexes in lumbar sympathetic neurons to increase bladdercapacity or elicit parasympathetic bladder efferent excitation totrigger a normal micturition reflex ( 8, 9, 11 ). However,the bladder capacity and/or the micturition reflex may be altered in anumber of pathophysiological conditions, such as interstitial cystitis, cyclophosphamide-induced cystitis, and irritant-inducedhypersensitivity ( 14 ). The mechanisms responsible forbladder hyperactivity may vary from condition to condition and arelikely complicated. Bladder biopsies in some patients diagnosed withinterstitial cystitis have shown increased density of SP-containingfibers and NK 1 receptors ( 15, 26, 29 ).Furthermore, administration of SP is known to cause bladderinflammation ( 1, 21, 39 ) and generation of reactive oxygenspecies (ROS) by inflammatory cells ( 3, 40 ). Thus SP mayhave a direct or an indirect role in neurogenic inflammation andregulation of bladder motility in various clinical conditions.Nonetheless, whether ROS generated by SP-reacted inflammatory cellscontribute to bladder hyperactivity remains to be determined." E7 p5 {/ U+ r# @0 h6 V

Our objective in the present study was to clarify the contribution andthe possible mechanism of SP in bladder hyperreflexia. We showed thatbladder hyperactivity caused by pelvic nerve stimulation is associatedwith an increased SP level in the bladder. We also used NK-receptorantagonists and free radical scavengers to test the role of SP- andROS-induced hyperactivity, respectively. Our results clearly showedthat SP via NK 1 -receptor activation enhances themicturition reflex and ROS release from the inflammatory cells and,consequently, leads to a hyperactive bladder.
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MATERIALS AND METHODS0 z3 }4 n4 S- |& A
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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 onthe day of the experiments.

Table 1. Drugs/chemicals% i. R3 c8 c. N! \
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Surgery. Adult female Wistar rats weighing 220-240 g were anesthetized withurethane (1.2 g/kg sc), which is well known to anesthetize the animalsyet permit full reflex bladder contractions ( 8, 9 ).Maintenance of deep anesthesia was determined by the persistence ofmiotic pupils as judged from frequent inspection and by the lack ofheart rate and arterial blood pressure (ABP) fluctuations in theabsence of visceral stimuli ( 8 ). An experiment wasterminated when the baseline mean ABP was protocol were in accordance with the guidelines of theNational Science Council of the Republic of China (1997). All effortswere made to minimize animal suffering and the number of animals used throughout the experiment. At the end of each experiment, the animalswere killed by an intravenous potassium chloride injection.

PE-50 catheters were placed in the left femoral artery for measurementof 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-10tubing inserted just above the bifurcation of the aorta from the rightfemoral artery was used for injection of various drugs. Bodytemperature was kept at 36.5-37°C by an infrared light and wasmonitored with a rectal thermometer.
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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 SPin the bladder micturition reflex and bladder hyperactivity ( 8, 18 ). Briefly, the urinary bladder was exposed through a midlineincision of the abdomen, and urine was emptied by application of lightpressure. A PE-50 T tube was inserted through the apex of the bladderdome. The bladders were filled by continuous infusion of 0.9% saline(0.15 ml/min) at room temperature and allowed to drain/micturaterepeatedly via the urethra.

The change in bladder pelvic afferent nerve activity (PANA), bladderpelvic efferent nerve activity (PENA), ABP, and various parametersmeasured by the cystometrogram can be determined before and afterintra-arterial or intrathecal administration of various chemicals. Allchemicals were injected through the intra-arterial catheter in a volumeof 1 ml/kg (0.20-0.25 ml) and were followed by 0.1 ml ofheparinized saline. For intrathecal administration, a catheter (Portex,Hythe, Kent, UK) was inserted through the atlantooccipital membrane for8.5 cm, such that the tip of the catheter was placed just above thelumbosacral enlargement, as described previously ( 18 ).Chemicals were given intrathecally in a volume of 20 µl and werefollowed by 30 µl of saline.- u& ~* z, L# E% I+ i

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 thebladder micturition reflex by administration of NK-receptor antagonists; groups 2 and 3 were used to test theeffect of exogenous SP and SP NK-receptor antagonists on bladderhyperactivity. 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 afterintra-arterial injection. To verify the SP activity via theNK 1 receptor, we injected SP ( group 2 ), SP the NK 1 -receptor antagonist CP-96345 ( group3a ), or the NK 2 -receptor antagonist SR-48968( group 3b ). The NK-receptor antagonists were given at250-500 µg ia and 5-250 µg it, doses that were lower thanthose used in previous studies ( 5,000 µg/kg iv and 250 µg/kgit) ( 17, 24, 27 ).

Cystometrogram for measurement of bladder response. The bladder catheter was connected via a T tube to a pressuretransducer (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 werealso measured: threshold volume (infused volume at the point precedinga micturition reflex), number of active 15 mmHg),micturition volume (volume of expelled urine collected in a preweighedtube), residual volume (amount of fluid remaining after a bladdercontraction; infused volume = infusion rate micturitionvolume), bladder capacity (residual volume micturition volume),intercontraction interval (time lag between 2 micturition cycles), andbasal pressures.' A2 f" _1 ?# Y

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 bladdersurface were isolated and simultaneously recorded by placement of theintact nerve fibers in parallel with two pairs of thin, bipolarstainless steel electrodes ( 8 ). The electrical signals were amplified 20,000-fold, filtered (high-frequency cutoff at 3,000 Hzand 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 awindow discriminator (model 121, World Precision Instruments, Sarasota,FL) and analyzed with an impulse counter (Gould integrator amplifier13-4615-70) that was set to count the total number of spikes per second( 8, 9 ). The background activity, which could be caused bythe nerve contact with electrodes, nerve damage during handling, andthe equipment itself, was excluded from the window discriminator byadjustment of the threshold voltage ( 9 )., g* @2 a$ @/ O1 O& Q, P+ a6 r

Nerve fiber with PANA was confirmed by its ability to show increasedactivity in response to small increments in IVP by saline infusion viaT tube. Nerve fiber with PENA had minimal activity until a thresholdvolume/pressure in the bladder produced a bursting discharge causing amicturition contraction (Fig. 1 )( 8, 9, 36 ).5 G( d) F7 }: D! a. s, |
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Fig. 1. Saline infusion (0.15 ml/min) into the urinary bladder evokingnormal micturition demonstrated with a transcystometric bladder model. A : simultaneous recordings of pelvic afferent nervousactivity (PANA) and pelvic efferent nervous activity (PENA),intravesical pressure (IVP), and arterial blood pressure (ABP) at aslow chart speed. B : at a fast chart speed, accumulatedurine in the urinary bladder gradually enhancesmechanoreceptor-dependent PANA. Up to a threshold volume, the enhancedPANA evokes a bursting type of PENA and triggers a series offluctuating bladder contractions to expel the urine. A mild increase inABP, defined as a vesicovascular reflex, is associated with amicturition reflex. N, neurogenic bladder contraction.

Electrical stimulation of the pelvic nerve. The next study was intended to determine whether nerve activity causingbladder hyperactivity was accompanied by release and/or elevation of SPin bladder. An electric current of square-wave pulses with pulseduration of 0.05 ms was applied from a stimulator (model S88, Grass,Quincy, MA) through a stimulus isolation unit (model SIU5B, Grass) anda constant-current unit (model CCU1A, Grass).

Six rats were given a solution of atropine (1 mg · ml 1 · kg 1 ia) toeffectively inhibit the muscarinic (parasympathetic) effect. One branchof the pelvic nerve was dissected from the left major pelvic ganglionto the urinary bladder and was identified by a bladder contractionelicited by electrical stimulation ( 8 ). The urinarybladder was electrically stimulated (1-10 Hz) for 5 min, andbladder response, including IVP and the number of active contractions,was recorded. Blood samples from the bladder outflow (iliac vein) wereobtained for determination of SP level before and during each period of stimulation.
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Measurement of SP. Plasma levels of SP in the iliac vein were measured as describedpreviously ( 5 ). The study was intended to determinewhether pelvic nerve activity results in elevation and release of SP in the bladder. Briefly, supernatant from plasma samples was diluted withthe same volume of buffer A (RIK-BA-1, PeninsulaLaboratory). Then each sample was passed slowly through a C18 Sep-Pakcolumn (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 byvacuum centrifugation and stored at 70°C for later analysis. An SPenzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) was used todetect the SP level. Each sample was dissolved in 1% HCl, diluted to asuitable concentration with enzyme immunoassay buffer, and assayed induplicate. The SP, which was linked to acetylcholinesterase as atracer, 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 plateswere read at 410 nm, and SP levels were calculated.' r5 Q; n1 w6 ~" P- c6 J6 F+ t/ @4 @( p

Detection of ROS production in urinary bladder after exogenous SPadministration. SP is capable of causing intracellular ROS generation and release byinflammatory 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. TheROS generation in response to SP stimulation was measured in wholeblood (obtained from the femoral artery) and bladder by achemiluminescence (CL) detection method as described previously ( 7 ). In addition, the possible cellular source of ROS inthe urinary bladder was examined by the fluorescence emitted after dichlorofluorescin (DCFH) diacetate infusion ( 38 ).& a/ M2 ^$ _- K2 Y9 G

A change in the bladder surface during the filling and micturitionstates of the transcystometric model may affect the measurement of ROS.Thus an isovolumetric model allowing minimal bladder surface change wasadopted for measurement of ROS generation from the bladder in vivo.Eighteen rats were used and grouped (see below) in this part of theexperiment. To establish an isovolumetric condition, we inserted onePE-50 tube into the bladder through the urethra and tied it in placewith a ligature around the urethral orifice ( 23 ). Thecatheter was connected to a separate pressure transducer and aninfusion pump via a T tube connector. Transurethral filling (0.15 ml/min) of 0.9% saline into the urinary bladder via the urethralcatheter was done until rhythmic bladder contractions occurred. Theinfusion was stopped, and the bladder was maintained underconstant-volume conditions by ligation of the ureter bilaterally.
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The method for detection of ROS from the organ surfaceafter2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-(1,2- a )-pyrazin-3-one hydrochloride administration (0.2 mg · ml 1 · h 1 ia) wasadapted from the technique described by Chien et al ( 7 ) for demonstration of ROS production in the hyperactive bladder. The ratwas maintained on a respirator (1.0-1.5 ml tidal volume, 80-90 cycles/min, 20-30 cmH 2 O inspiratorypressure) and a circulating water pad at 37°C during photondetection. For exclusion of photon emission from sources other than theurinary bladder, the animal was housed in a dark box with a shieldedplate. Only the bladder window was left unshielded and was positionedunder a reflector, which reflected the photons from the exposed bladdersurface onto the detector area. The2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-(1,2- a )-pyrazin-3-one hydrochloride-enhanced CL signal from the bladder surface was measured continuously before and during SP (10 µg) administration byuse of a CL analyzing system (CLD-110, Tohoku Electronic Industrial, Sendai, Japan).5 W/ x, R$ H3 p  H+ G& y

Cellular origin of ROS in the bladder after SP administration. We conducted DCFH diacetate tissue staining to determine the cellularorigin of ROS in the bladder after SP administration. DCFH diacetate isa stable nonfluorescent compound that can diffuse into cells, ishydrolyzed to DCFH, and is thereby trapped within the cells( 38 ). DCFH is oxidized by ROS to yield dichlorofluorescein (DCF), a fluorescent molecule ( 38 ).( l' }: O4 m& J1 \8 a6 ?# w
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At 10 min after SP (10 µg ia) administration, the bladder wasfilled 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 optimalcutting temperature compound (Sakura Finetek, Torrance, CA). Frozensections (10 µm thick) were obtained, and cellular fluorescenceintensity in the tissue sections was examined by fluorescencemicroscopy (model DMRD, Leica Microsystems Wetzlar, Wetzlar, Germany).
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In addition, a portion of the urinary bladder was cut and fixed in 10%neutral buffered formalin solution, dehydrated in graded ethanol, andembedded in paraffin. Sections (5 µm) of bladders were stained withhematoxylin and eosin and Giemsa and evaluated for the extent ofinflammatory cell accumulation and number of mast cells( 31 ).! A4 R+ W( g0 n7 d; }! _
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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 bladderhyperactivity. Before SP stimulation, baseline ABP, IVP, and ROSamounts were recorded for 30 min as a control value. After 10 min of SPstimulation, the rats ( n = 18) were divided into groupsand treated with saline ( n = 2), CP-96345 (500 µg, n = 4), SR-48968 (500 µg, n = 2),superoxide dismutase (SOD, 500 U, n = 4),FC 4 S (250 µg, n = 3), or C 3 (250 µg, n = 3). The three antioxidants (SOD and thefullerenes FC 4 S and C 3 ) were used to testwhether the SP-induced bladder hyperactivity can be ameliorated by freeradical scavengers. The ABP and cystometrogram were monitored simultaneously for 60 min. After measurement, the bladder tissues andleukocytes isolated from 4 ml of whole blood were collected and storedat 70°C for immunoblotting analysis.2 R2 I  R6 t" j! Q4 M' Z$ v

Immunoblot analysis for ICAM and -actin. The immunoblotting method was described previously ( 7 ). Wemeasured the amounts of ICAM and -actin in bladder tissues andleukocytes of SP-treated rats. For protein analysis, bladder samplesand leukocytes were homogenized with a prechilled mortar and pestle inextraction buffer, which consisted of 10 mM Tris · HCl (pH7.6), 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% NP-40, 0.5辭xycholate, 2% -mercaptoethanol, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin. The mixtures were homogenized completely byvortexing and kept at 4°C for 30 min. The homogenate was centrifuged at 12,000 g for 12 min at 4°C, the supernatant wascollected, and protein concentration was determined by Bio-Rad proteinassay (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.5 j% {2 t, V  F. l- N9 ?
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SDS-PAGE was performed on 12.5% separation gels in the absence ofurea, and the gels were stained with Coomassie brilliant blue. Proteinson the SDS-PAGE gels, each lane containing 30 µg of total protein,were transferred to nitrocellulose filters. The immunoreactive bandswere detected by incubation with the antibody described above, thesecondary antibody-alkaline phosphatase, and, finally, nitro bluetetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt(Roche Diagnostic, Mannheim, Germany) stock solution for 30 min at room temperature.4 A8 R0 w+ s# z  ^

Data acquisition and statistical analysis. All nervous activity (PANA and PENA) was expressed as the number ofspikes per second. We analyzed the PANA and the frequency of burstingPENA before and after drug treatment. Values are means ± SE. Datawere subjected to analysis of variance, followed by Duncan'smultiple-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 indicate statistical significance.- H9 N9 n4 x& Z+ \$ G$ q) w

RESULTS9 m7 [# n7 f5 z5 G' d2 m0 l

The PANA and PENA, IVP, and ABP in rats with normal micturitionreflex were recorded simultaneously. The IVP was gradually increased onaccumulation of saline in the urinary bladder, causing activation ofPANA. The frequency of PANA over time was progressively increased bythe 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 abursting efferent discharge (PENA) that lasted for 7-15 s wasdetected (Fig. 1 ). The bursting PENA can simultaneously trigger anactive neural reflex-mediated bladder contraction, as shown by a quickrise in IVP. ABP was slightly and transiently elevated (for~15-35 s) in accordance with the abrupt rise in IVP and theneurogenic bladder contraction (Fig. 1 ).
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Endogenous SP via NK 1 receptor participates in a normalmicturition reflex. Intra-arterial administration of CP-96345 (250 µg) significantlydecreased the frequency of PANA over time, the frequency of burstingPENA, and active contractions ( P bladder capacity (Fig. 2, Table 2; P that intra-arterial CP-96345 exerts apartial inhibition on afferent neurotransmission of the micturitionreflex to increase the bladder capacity. A more significant effect ofCP-96345 was detected when it was administered intrathecally. The meanfrequency of PANA over time increased after intrathecal CP-96345administration, while the peak frequency of PENA dropped to zero,indicating that lumbosacral spinal NK 1 -receptor blockadecompletely inhibited the afferent and efferent neural transmission ofthe micturition reflex. Inhibition in micturition parameters isdisplayed as a function of dose (Fig. 3,Table 2 ). The abrupt and dose-dependent hypotensive response tointrathecal administration of the NK 1 antagonist indicatesCP-96345 leakage into the systemic circulation. In contrast,intra-arterial or intrathecal administration of SR-48968 had noinfluence on the micturition reflex (Table 2 ), suggesting that SP viamainly the NK 1, not the NK 2, receptor mediatesthe afferent micturition pathway in the lumbosacral spinal cord (Fig. 2, right, and Fig. 3 C ).8 R, T3 ~9 X( _! t7 I0 R' m- j1 d

Fig. 2. Effects of intra-arterial CP-96345 and SR-48968 on micturitionparameters. The bladder was continuously filled with normal saline at0.1 ml/min. The frequency of PANA over time was progressively increasedby the increase of bladder filling volume. When a threshold volume wasreached, the frequency of PANA over time reached its maximal value, andthen a peak frequency of PENA and a sharp rise in IVP were detected. Left : CP-96345 increased the intercontraction interval(i.e., decreased the frequency of active contractions) and decreasedthe peak frequency of PENA. Right : intra-arterialadministration of SR-48968 (250 µg) did not influence any parametersof the micturition reflex. *Significant elongation of intercontractioninterval compared with control.+ g0 O1 T  b8 j+ A" F# i
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Table 2. Effects of intra-arterial or intrathecal administration of SP,CP-96345, and SR-48968 on micturition parameters inurethane-anesthetized rats" k( N; N, D5 [  K6 F- X1 @

Fig. 3. Inhibitory effects of intrathecally administered low( A, left ), medium ( A, right ), or high( B ) dose of CP-96345 on micturition parameters. Bladdercontraction was induced by continuous infusion of saline into thebladder. Intrathecally administered CP-96345 markedly depressedthe peak frequency of PENA-mediated micturition, although the frequencyof PANA was increased over time by the overdistended bladder. Becauseof inhibition of peak frequency of bursting PENA-mediated micturitionand of accumulated urine volume, a prolonged and enhanced frequency ofPANA over time occurred in the overdistended bladder ( A,right and B ). The inhibitory effect of CP-96345 on themicturition reflex was dose dependent. Intrathecally administeredSR-48968 ( C ) had no effect on micturition parameters.
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Pelvic nerve stimulation enhances bladder hyperactivity and SPrelease. In rats, an increase in frequency (1-10 Hz) of pelvic nervestimulation 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 SPto 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 bladderhyperactivity caused by pelvic nerve stimulation is associated with adose-dependent increase in SP in the bladder. Furthermore, we suggestthat, in addition to SP, NKA and other substances are released fromsensory pelvic afferents during electrical stimulation and,consequently, lead to a prolonged bladder contraction.

Fig. 4. Effect of pelvic nerve stimulation on bladder activityand plasma substance P (SP) concentration from the bladder outflow. A : graded increases in frequency of pelvic nerve stimulationincreased the duration of bladder contractions and decreased theintercontraction intervals. B : increased frequency ofelectrical stimulation also enhanced SP release into the iliac vein.* P

Exogenous SP facilitates the micturition reflex in bladderhyperreflexia. At residual volume immediately after micturition, no significant PANAand PENA were observed, and the IVP remained at the basal level whensaline infusion was stopped (Fig. 5 A ). At ~5-10 s afterintra-arterial administration of SP (0.1-5 µg), the bladder revealed an elevated IVP and dose-dependently enhanced PANA and PENA(Fig. 5 B, fast trace ).

Fig. 5. Excitatory effects of intra-arterial SP on micturitionparameters at residual volume. A : at residual volume withoutsaline infusion, SP increased PANA, PENA, and IVP and lowered ABP in adose-dependent manner. B : SP triggered myogenic (M) bladdercontraction and PANA- and PENA-mediated neural (N) bladder contraction. C : at residual volume with continuous saline infusion, SPproduced a tonic contraction followed, during its relaxation phase, bya series of rhythmic contractions (micturition reflex), which persistedeven when the tonic contraction had returned to resting values. Noteincreased frequency of micturition (or shortened intercontractioninterval) after SP stimulation.

During filling, the bladder revealed increased PANA by mechanicaldistension of bladder afferents. Shortly after intra-arterial administration of SP (10 µg), a bursting PENA was followed by aprolonged (~10-min) tonic bladder contraction (Fig. 5 C ).At 10 min after SP stimulation, the bladder remained hyperactive, asshown by the increase in the frequency of active contractions (numberof active contractions; P bladder capacity ( P 2 ). Our resultsfurther indicated that intra-arterial administration of SP can initiatethe micturition reflex response in bladders containing a subthresholdamount (residual volume) of fluid, e.g., by activating NK receptors insmooth muscle and sensory neurons and by increasing the afferentdischarge (PANA) to the central nervous system to result in ahyperactive bladder. A similar result was reported previously( 23 ).

Similarly, intrathecal SP administration also induced bladderhyperactivity (Fig. 6 ), as evidenced byan increase in the number of active contractions, a decrease in bladdercapacity, and an elevation of baseline pressure (Table 2 ). Theincreased baseline pressure may be due to an increased release oftachykinins and other substances by continuous motor impulses to thesmooth muscle through the pelvic parasympathetic nerves, which isbrought about by micturition reflex afferents. These data suggest thatincreased SP in the rat lumbosacral spinal cord also plays a role ininducing bladder hypersensitivity via an afferent pathway.

Fig. 6. Effects of intrathecal saline and SP on micturition parameters.Bladder contraction was induced by continuous infusion of saline intothe bladder. Intrathecal saline had no effects on micturitionparameters. Intrathecal SP evoked a systemic vasodilation and ahyperactive bladder, including a high frequency of IVP, PANA, and PENAand an elevation of baseline pressure.  c. b* X4 t/ C: ^  ~/ v

Increased ROS formation from whole blood and bladder surface afterexogenous SP administration. The SP-treated whole blood samples showed a dose-dependent increase inROS formation: 78 ± 20, 336 ± 67, 560 ± 105, 1,035 ± 213, 1,201 ± 196, and 1,108 ± 230 counts/10 sat 0, 0.1, 1, 10, 50, and 100 µg of SP, respectively (Fig. 7 A ). The source of whole bloodROS was leukocytes, not erythrocytes and plasma (Fig. 7 C ).Coincubation with the NK 1 -receptor antagonist CP-96345 (50 µg) prevented SP-induced ROS generation from whole blood, which wasreduced from 1,065 ± 210 to 97 ± 18 counts/10 s in response to treatment with 50 µg of SP (Fig. 7 B ). In contrast,SR-48968 had no significant inhibitory effect on SP-induced ROSgeneration: 1,065 ± 210 vs. 899 ± 177 counts/10s ( P 0.05). Coincubation with the free radicalscavengers SOD, C 3, and FC 4 S also significantly reduced the SP-induced ROS generation: 220 ± 51, 430 ± 89, and 190 ± 56 counts/10 s, respectively ( P the reduction exhibited by free radicalscavengers was slightly lower than that exhibited by CP-96345,suggesting that directly blocking upstream NK 1 -receptoractivation by a long-lasting effect of the nonpeptide CP-96345 is moreefficient than scavenging downstream ROS activity by free radicalscavengers.5 l. C# C1 q5 I2 b# @, C

Fig. 7. SP-induced reactive oxygen species (ROS) generation in whole blood. A : 0-100 µg of SP induced whole blood ROS formationin a dose-dependent manner, with maximal ROS formation at 50 µg. B : primary sources of ROS were leukocytes (WBC), noterythrocytes (RBC) or plasma. C : effect of 50 µg of SP canbe prevented by coincubation with CP-96345, SOD, C 3, andFC 4 S, but not SR-48968. D : whole blood ROS fromrats significantly increased with intra-arterial SP treatment (10 µg/rat in 0.2 ml of saline). These enhanced ROS can be reducedsomewhat by simultaneous treatment with CP-96345, SOD, C 3,or FC 4 S ( D ), but not SR-48968. * P # P* R0 j- N! e0 X! h( Z+ t

Blood samples from rats subjected to intra-arterial SP treatment wereexamined for ROS generation. ROS in whole blood increased significantlyfrom 101 ± 18 to 2,198 ± 365 counts/10 s after SP stimulation (Fig. 7 D ). This increase was reduced somewhat bysimultaneous treatment with CP-96345 (756 ± 134 counts/10 s), SOD(897 ± 168 counts/10 s), C 3 (1,312 ± 260 counts/10 s), or FC 4 S (1,032 ± 185 counts/10 s), butnot by SR-48968 (2,250 ± 368 counts/10 s).9 ]) y5 x: ?5 T: g8 w

Inhibition of bladder hyperactivity by free radical scavengers. In the isovolumetric condition without SP stimulation, the frequency ofbladder 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 the frequency of active contractions (7.3 ± 1.7 active contractions/10 min, P 8 A; from140 ± 25 counts/10 s before SP stimulation to 2,560 ± 345 counts/10 s after 10 min of SP, P intra-arterial saline had no effect on theSP-induced response. The response of the hyperactive bladder and theincrease in ROS 30 min.9 i9 b' M* [/ \: m
# r* |+ V% g6 y( f( z# m% n
Fig. 8. In vivo response of SP-induced bladder hyperactivity andbladder ROS generation in an isovolumetric model. A :intra-arterial SP injection induced significant vasodilation andbladder hyperactivity, as well as bladder ROS generation. A and B : saline or SR-48968 had no effect on SP-inducedbladder hyperactivity and ROS production. C : treatment withCP-96345 reduced the frequency of micturition and decreased, in part,ROS generation in the bladder ( right ). D :superoxide dismutase (SOD) injection decreased ROS generation in thebladder and reduced the frequency of micturition ( right ).* ^: X5 F7 a3 O6 d7 k
* i' j' m- L) m5 i
Pretreatment of NK-receptor antagonist is well known to reducetachykinin-induced hyperactivity ( 18, 19, 21, 22 ); therefore, for evaluation of the possibly pharmacotherapeutic potentialof agents on SP-induced hyperactivity, we administered the test agents10 min after SP stimulation. At 10 min after SP stimulation,intra-arterial administration of CP-96345 significantly decreased thefrequency of bladder contractions (to 3.4 ± 0.8 activecontractions/10 min, P generation from the bladder surface (Fig. 8 C, right; from2,705 ± 453 to 1,266 ± 320 counts/10 s, P of SOD (Fig. 8 D,right ), C 3, and FC 4 S also significantlydecreased the frequency of bladder contractions (to 4.5 ± 1.1, 4.9 ± 1.2, and 4.2 ± 1.0 active contractions/10 min withSOD, C 3, and FC 4 S, respectively) and ROSgeneration (to 1,556 ± 345, 1,956 ± 455, and 1,705 ± 345 counts/10 s with SOD, C 3, and FC 4 S,respectively). However, SR-48968 had no effects on SP-inducedhyperactivity and ROS production (Fig. 8 B ). These datashowed that ROS plays a role in SP-induced bladder hyperactivity./ y- X0 u% i3 D6 t/ A" ]# N
  ^% T8 H, o3 _5 F4 f4 R6 ~
Cellular origin of ROS in SP-induced bladder inflammation. Sections from the bladder mucosa and smooth muscle obtained40-45 min after SP administration revealed infiltration ofleukocytes and the presence of several degranulated mast cells (Fig. 9 ). Sections of urinary bladders fromcontrol animals exhibited no signs of inflammation at the end of theexperiments." ~, K# k1 k+ S& c+ b

Fig. 9. Mast cells (arrows) and leukocytes (pluses) in control( A ) and SP-induced inflamed ( B ) urinary bladder.Magnification ×400. Mast cells and leukocytes primarily occurred inthe vicinity of the blood vessels in the SP-treated bladder, but not inthe control bladder. C and D : some leukocytesadhered to the endothelium of one vessel, where mast cell degranulationwas found. Dichlorofluorescin diacetate and propidium iodide under afluorescence microscope were used for localization of ROS productionand cell viability in bladder tissue. Green fluorescence ofdichlorofluorescein was apparent in vessels ( F ) andsubmucosal layer ( G and H ), where numerous cellswith red fluorescence (propidium iodide staining) were found( G and H ), suggesting oxidative damage. Neithergreen nor red fluorescence was observed in the control bladder( E ).$ E( F) z3 u9 n5 g

We used DCFH diacetate and PI under a fluorescence microscope forlocalization of ROS production and cell viability in bladder tissue,respectively. We found green fluorescence of DCF mainly in neutrophilsin and around vessels (Fig. 9 F ) and in the submucosal layer(Fig. 9, G and H ) of the bladder. In a similarlocation of the green fluorescence of DCF, red fluorescence of PI wasobserved (Fig. 9, F-H ), indicating the presence ofROS-induced cellular damage. Neither green DCF fluorescence nor red PIfluorescence was observed in the control bladders (Fig. 9 E ).
; L; m; d3 x9 l4 S
Effect of NK-receptor antagonist or SOD on ICAM expression inSP-treated bladder and leukocytes. Expression of ICAM and -actin in the bladder and leukocytes after SPtreatment was assessed by immunoblotting with antibodies against ICAMand -actin (Fig. 10 ). ICAMexpression was detected in control bladder and leukocytes. ICAMexpression in bladder tissues was dose dependently increased by SPstimulation: 1.0 ± 0-, 2.0 ± 0.3-, and 3.5 ± 0.5-foldfor 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 enhancedICAM expression (3.5 ± 0.5- vs. 3.1 ± 0.6-fold). ICAMexpression in leukocytes was unaffected by SP stimulation.6 B! u# Y8 y1 ^( R$ b; q
! ]: n1 O* `0 e3 V3 }
Fig. 10. Top : Western blot analysis of homogenates ofrat bladders and leukocytes subjected to SP stimulation with specificantibodies to intercellular adhesion molecule (ICAM) and -actin.Note dose-dependent increase in ICAM (85 kDa) expression after SP (1 and 10 µg) stimulation. ICAM expression appeared to be decreasedafter treatment with CP-96345 (CP, 500 µg) and SOD (500 µg), butnot SR-48968 (SR, 500 µg). In leukocytes, SP had no effect on ICAMexpression. Equal protein loading was displayed by -actin. Bottom : mean densitometric data. * P # P4 a1 C" \- Z# ~. Z7 I

DISCUSSION
% [# k! ~! F: o5 l0 J. v; C8 V
The present study demonstrates for the first time the involvementof ROS generation in SP-mediated bladder hyperreflexia. SP is a sensoryneuropeptide present in small myelinated A -fibers and insmall-diameter unmyelinated C fibers ( 22 ). Once released from bladder afferent nerves and the sacral spinal cord, SP is involvedin the mechanoreceptor-mediated micturition reflex. In rats, systemicadministration of capsaicin for depletion of SP resulted in urineretention or an increased volume/pressure threshold for micturition,implicating an excitatory role of SP in the afferent micturitionpathway ( 6, 22 ). We demonstrated that intra-arterial administration of exogenous SP could initiate the micturition reflexresponse in bladders containing a subthreshold amount (residual volume)of fluid. The SP-mediated micturition reflex or bladder hyperactivityis mainly through NK 1 receptors. We further showed thatexogenous or excessive SP stimulation also resulted in increased ROSgeneration. NK 1 -receptor blockade or free radicalscavengers could inhibit micturition pathway facilitation andSP-mediated ROS generation and, subsequently, ameliorate SP-inducedbladder hyperactivity.: p0 K0 ~- G# p$ m- v' G
7 |8 o" \! ~+ x8 T9 L
In our study, the cystometrogram and electrophysiological recordingtechniques in urethane-anesthetized rats are valuable tools foranalyzing the neural control of the urinary bladder ( 6, 9, 36 ). Efficient bladder voiding is triggered primarily by thecumulative afferent activities from the bladder mechanoreceptors duringbladder distension, which subsequently elicits parasympathetic bladderefferent excitation to evoke a micturition reflex when a thresholdvolume/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) offluid. A normal bladder contraction is coordinated by efferentpurinergic, 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 innormal micturition and bladder hyperactivity remain to be furtherelucidated ( 21 ).9 a- g! N8 a# n' K, q
/ g9 |- [  m; {  ]
Stimulation by exogenous SP or NK-receptor agonists has been shown toinduce bladder hyperactivity ( 18, 21, 22 ). Maggi ( 22 ) demonstrated facilitation of reflex micturition byintravesical administration of [ -Ala 8 ]-NKA (anNK 2 -receptor agonist), suggesting a peripheral site ofaction via smooth muscle contraction in the bladder. SP can also be acentrally excitatory neurotransmitter producing a slow excitatorypostsynaptic potential in the neurons of the spinal cord and mediatinga dorsal root C fiber reflex to the dorsal horn neurons, leading tobladder hyperreflexia (Fig. 3, Table 2 ). Thus SP exerts effects on theperipheral bladder and the central spinal cord to facilitate themicturition pathway and, consequently, lead to bladder hyperactivity.The SP-mediated hyperactivity can be inhibited by intra-arterial orintrathecal administration of CP-96345 (an NK 1 -receptorantagonist), but not SR-48968 (an NK 2 -receptor antagonist).

It is also known that a peripheral release of tachykinins determines aset of responses (loosely defined as neurogenic inflammation) thatincludes vasodilatation, plasma protein extravasation, smooth musclecontraction, stimulation of afferents, and inflammation ( 19 ). To further complicate matters, sensory neurons andimmune cells can express and release tachykinins, which may alsocontribute to neurogenic inflammation in the bladder. SP (0.3-1µM) is able to induce, in a dose-dependent manner, secretion ofvarious cytokines (e.g., interleukins-1 and -6 and tumor necrosisfactor- ) from cultured lymphocyte-enriched mononuclear cellsisolated from human peripheral blood ( 10 ). In addition, SPhas been shown to cause a proinflammatory change in tissues, such asdegranulation of mast cells and leukocyte adhesion to the venularendothelium ( 39 ), by a mechanism of SP-enhanced ICAMexpression in the bladder tissue (Fig. 10 ). The possible involvement ofSP and its NK 1 receptor in pathophysiological changes ofbladder inflammation has been underscored by a recent study showing adramatic reduction in antigen-induced cystitis inNK 1 -receptor-deficient mice ( 31 ).9 f0 x8 N& ^# }3 ~1 a' }0 B* L
4 d& O& E3 f1 p4 X; \& J* z& I
As a consequence of SP-immune and -inflammatory cell interaction, avariety of substances, such as histamine, cytokines, and ROS, arereleased ( 3, 10, 32, 39 ). In whole blood and leukocytesincubated with SP, ROS activity was displayed in a dose-dependentmanner and SP-induced ROS release and ICAM expression are inhibited byCP-96345, but not SR-48968, confirming a mediating role of theNK 1 receptor in triggering ROS generation in leukocytes. Wefurther showed that SP-induced ROS generation in the bladder as well asbladder hyperreflexia can be partly ameliorated by CP-96345 or,noticeably, by free radical scavengers. It is not totally unexpectedthat SP-induced ROS formation may contribute to the myogenic and neuralhyperactivity. ROS are known to be involved in changes in muscle tone,vascular smooth muscle strip contraction ( 2, 30 ), andincreased neural activity/conduction velocity in vitro by mechanismssuch as alterations in membrane conductance, calcium homeostasis,calcium-dependent processes, and eicosanoid and nitric oxide metabolism( 2, 16, 42 ).* U0 D" D0 n: b( {7 X8 N

In summary, our studies provided direct evidence that SP participatesin the micturition reflex response in bladders by activating NKreceptors to facilitate the afferent pathway. Increased SP stimulationmay enhance the afferent discharge (PANA) to the central nervous systemto result in a hyperactive bladder. On the other hand, increased nerveactivity by other means may result in increased release of SP, furthercomplicating bladder hyperactivity. Our study indicates that themechanism by which SP participates in the neurogenic bladder may becomplicated by its proinflammatory activity and its ability tostimulate ROS generation.4 v9 h$ z$ V6 ?9 {6 D- `

ACKNOWLEDGEMENTS
. T2 E7 A4 I  B, ~
This work was supported in part by National Taiwan UniversityHospital Grants NTUH89A014, 89A023-10, and 89A023-11, National ScienceCouncil of the Republic of China Grants NSC90-2320-B-002-035, NSC90-2320-B002-152, NSC90-2314-B-002-446, andNSC89-2314-B-002-350, and the Mrs. Hsiu-Chin Lee Kidney ResearchFund (to C.-T. Chien).
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