Vanilloid receptor and detrusor instability

VANILLOID RECEPTOR AND DETRUSOR INSTABILITY

FRANCISCO CRUZ

ABSTRACTVery recently, a membrane receptor (vanilloid receptor type 1 [VR-1]) sensitive to capsaicin or resiniferatoxin(RTX) was identified in small- and medium-sized dorsal root ganglion neurons that give rise to mostunmyelinated sensory fibers. After vanilloid binding to VR-1, these neurons remain transiently desensitized;that is, less reactive to natural stimuli. It is this effect of vanilloid substances that is being investigated for itspotential therapeutic utility. In the urinary bladder, VR-1–expressing fibers are extremely abundant in themucosa and in the muscular layer. In the latter, VR-1 fibers are intimately apposed to smooth muscle cells.The demonstration, several years ago, that these fibers were involved in detrusor hyperreflexia of spinalorigin and in bladder pain processing, justified the clinical application of intravesical capsaicin or RTX inhumans with these bladder diseases. More recently, the experimental and clinical evidence that the sametype of bladder sensory fibers were also involved in detrusor instability made a strong case for intravesicalRTX assay in patients with idiopathic detrusor instability. UROLOGY 59 (Suppl 5A): 51–60, 2002. © 2002,Elsevier Science Inc.

In 1955, when Nicholas Jancso first made thefortuitous observation that nerve endings sub-

serving nociception could be activated and subse-quently inactivated by capsaicin, he could hardlypredict the future expansion of this field. It tookalmost 10 years before his study was accepted forpublication to a worldwide scientific audience.1Another decade had to pass before a specific neu-rotoxic action of capsaicin on small- and medium-sized dorsal root ganglion (DRG) neurons was of-fered as a putative explanation for those findings.2

During the last few years, an increasing numberof landmark publications have built on Jancso’sfindings. After descriptions of resiniferatoxin(RTX), an ultrapotent capsaicin analog extractedfrom the dry latex of the Euphorbia resinifera,3,4

and capsazepine, the first competitive antagonist ofcapsaicin5) specific binding sites for this substancewere autoradiographically visualized with [3H]RTXin primary sensory neurons of humans and otherspecies.4,6 More recently, using a molecular biol-ogy approach, the rat capsaicin–binding sites were

identified as a membrane protein called vanilloidreceptor type 1 (VR-1), and its gene, largely ex-pressed in small- and medium-sized DRG neurons,was cloned.7 A human ortholog of the rat VR-1gene was identified on chromosome 17.8

THE VANILLOID RECEPTOR

The rat VR-1 (rVR-1) is a membrane protein con-taining 838 amino acids. It has an N-terminus with3 ankyrin domains and a C-terminus without anyrecognizable motif. The rVR-1 is predicted to have6 transmembrane domains, forming a pore loopbetween the fifth and the sixth, and 3 protein ki-nase A phosphorylation sites.7 The human VR-1(hVR-1) is a sequence of 839 amino acids with 92%homology with the rVR-1. The highest homologyis seen in the region of the transmembrane do-mains and phosphorylation sites, whereas the low-est occurs in the N- and C-terminal sequences.8

The rVR-1 and hVR-1 are nonselective cationchannels. Activation of these channels in Xenopuslaevis oocytes by capsaicin induces massive Ca2�

and Na� inward currents that can be completelyblocked by capsazepine.7–10 These currents are,therefore, very similar to those evoked by capsaicinin rat DRG neurons maintained in culture.11 Asexpected from pharmacologic studies, RTX hashigh affinity for rVR-1.12 However, whereas inwardcurrents evoked by capsaicin are strong in ampli-tude but short lasting, those evoked by RTX are

From the Department of Urology, Hospital de S. Joao, Porto,Portugal; and Faculty of Medicine of Porto, Porto, Portugal

This work was funded in part by the Portuguese governmentthrough Fundacao Ciencia Tecnologia, Project POCTI/FEDER32466/NSE/00

Reprint requests: Francisco Cruz, MD, PhD, Hospital S. Joao,Department of Urology, Alameda Hernani Monteiro, 4200 Porto,Portugal. E-mail: [email protected]

© 2002, ELSEVIER SCIENCE INC. 0090-4295/02/$22.00ALL RIGHTS RESERVED PII S0090-4295(01)01638-7 51

weak yet long lasting,4,7 explaining the much highercalcium uptake induced by capsaicin over RTX.13

In addition to vanilloids, rVR-1 and hVR-1 areactivated by temperatures �43°C or by pH�5.7,8,14 At lower concentrations, protons, al-though being unable to open the receptor, enhancethe response to capsaicin and lower the heat thresh-old to room temperature.8,14 Inflammatory mediatorsalso enhance capsaicin-evoked inward currents incultured rat DRG neurons, although it remains un-clear whether they act directly on VR-1 or on second-order intracellular messengers.15 Altogether, thesedata make VR-1 the first receptor that can be viewedas an integrator of several painful chemical and phys-ical stimuli and a potential target for therapeutic in-tervention. This is reinforced by the recent observa-tion that VR-1 knockout rats are less reactive thancontrols to chemicals and heat.16

The consequences of vanilloid receptor activa-tion in sensory neurons include excitation, desen-sitization, and cell death. Cation influx that followsvanilloid receptor opening may cause membranedepolarization, which, after reaching the thresholdlevel, generates an action potential that can propa-gate centrally through the axon, giving rise to pain-ful or itch sensations.17,18 Vanilloids also induce amassive peripheral release of substance P and cal-citonin gene-related peptide (CGRP), possibly as aconsequence of voltage-sensitive Na� channel ac-tivation by antidromic propagation of action po-tentials, or by calcium-induced direct fusion ofneuropeptide-containing synaptic vesicles withthe neuronal membrane.19

Desensitization may follow VR-1 excitation byvanilloid compounds but not by other agonists,such as protons, heat, or proinflammatory media-tors. After repeated brief applications of capsaicinto sensitive neurons, desensitization causes a pro-gressive decrease in the amplitude of the inwardcurrents.7,17,20 The reduction in the bioelectricalactivity is accompanied by a decreased responsive-ness of the sensory neurons to natural stimuli, asshown by an increased threshold to noxious chem-ical and thermal stimuli.21–23 It is, therefore, desen-sitization, and not excitation, that offers potentialfor clinical application.

In sufficient concentrations, vanilloids may in-duce neuronal death by �1 mechanisms. Highintracellular levels of Ca2� and Na� that are gen-erated by vanilloid receptor opening may disruptcritical metabolic pathways in sensory fibers.24 Va-nilloids may deprive sensory fibers of crucial neu-rotrophic factors that are synthesized in peripheraltissues and normally transported through the ax-ons to the primary afferent cell bodies in theDRGs.25 Indeed, primary afferent degeneration af-ter administration of high doses of systemic capsa-icin to newborn rats can be partially prevented by

systemic administration of nerve growth factor(NGF).26 In addition, high intracellular concentra-tion of cations may cause severe osmotic swelling.7

INTERACTION OF THE VANILLOIDCOMPOUNDS AND VANILLOID RECEPTOR

TYPE 1Capsaicin and RTX are the most studied natural

compounds that bind to the vanilloid receptor. Theonly resemblance between the 2 molecules, how-ever, is the occurrence of an identical homovanillylring. Despite that, the role of this moiety in boththe initial excitation and the ensuing desensitiza-tion induced by these compounds is undeter-mined. Olvanil, a synthetic compound with potentdesensitizing properties, is devoid of acute excitingproperties, despite having a homovanillyl ring.27

On the other hand, neither polygodial (responsiblefor the pungency of water pepper, widely used inChinese and Japanese cuisine) nor scutigeral (ex-tracted from an edible, nonpungent mushroom)possess a homovanillyl ring.28 Similarly, anandam-ide, a lipid with some similarities to the long chainof the capsaicin or olvanil molecules but devoid ofa homovanillyl ring also evokes inward currents inrVR-129 and hVR-1.30 This is an intriguing obser-vation. Because anandamide is also a potent ago-nist of the cannabinoid receptor subtype-1, it raisesthe possibility of an interaction between vanilloidand cannabinoid receptors. On the other hand, be-cause anandamide is synthesized in brain tissue,31

vascular endothelium,32 and macrophages,33 it wassuggested that it may constitute another endoge-nous ligand for VR-1.

DISTRIBUTION OF THE VANILLOIDRECEPTOR IN THE URINARY BLADDER

An antibody against the N-terminus of therVR-1, produced by the Novartis Institute of Sci-ence (London, United Kingdom) has allowed theidentification of VR-1–immunoreactive (IR) vari-cose fibers in the rat urinary bladder (Avelino,Cruz, Nagy, and Cruz, unpublished data, 2000). Inthe mucosa, they form a loose network near thebasement membrane or between the epithelial cells(Figure 1A). In the muscular layer where they aremore numerous, VR-1–IR fibers course intimatelyapposed to smooth muscle cells (Figure 1B), sepa-rated from them by an empty narrow cleft whenexamined under the electron microscope (Figure2). Taking into consideration the known agonistsof the receptor, VR-1–containing fibers occurringin the mucosa seem particularly adequate to en-code pH and temperature changes. However, it isreasonable to assume that the coupling betweenVR-1 fibers and smooth muscle cells may addition-ally serve as a mechanoreceptor that encodes theirdegree of tension. This neuromuscular coupling

52 UROLOGY 59 (Supplement 5A), May 2002

may also facilitate the modulation of bladder con-tractility by sensory neuropeptides released fromprimary afferent endings.34

In DRG cells, VR-1 is equally expressed in pep-tidergic and nonpeptidergic primary afferent neu-rons, the latter being easily recognized by their af-finity to isolectin B4 (IB4).35–37 VR-1–IR bladderprimary afferents diverge, however, from this gen-eral distribution. Using the confocal microscope,substance P and CGRP immunoreactivity is foundin �90% of the VR-1–IR fibers coursing in thebladder wall. On the other hand, IB4-binding fiberscould not be identified in the rat urinary bladder(Avelino, Cruz, Nagy, and Cruz, unpublished data,2001).

The reason for the relative abundance of sensory

neuropeptides in VR-1 bladder sensory fibers, alsoconfirmed in electron micrographs by their rich-ness in large dense-cored synaptic vesicles (Figure2), is not yet clear. However, some physiologic im-plications may already be foreseen. Whereas pep-tidergic sensory fibers are sensitive to NGF, IB4-binding fibers are sensitive to glial cell–derivedneurotrophic factor.37 Thus, bladder afferents maybe particularly prone to an excess of NGF. Inter-estingly, in chronically obstructed rat bladders,possibly because of an excessive production ofNGF,38 bladder C-afferent cell bodies hypertrophy,and their spinal endings sprout around sacral para-sympathetic preganglionic neurons.39 This is ac-companied by their easy recruitment during pelvicnerve stimulation.40 In addition, because VR-1–

FIGURE 1. (A) Vanilloid receptor type 1–immunoreactive (VR-1–IR) fibers in the bladder mucosa are close to orbetween the epithelial cells. (B) VR-1–IR fibers in the muscular layer run parallel to the smooth muscle fibersintimately apposed to their surface.

UROLOGY 59 (Supplement 5A), May 2002 53

mediated currents in IB4-binding and peptidergiccells are different,41 the paucity of IB4-binding fi-bers among VR-1–IR bladder afferents may also in-dicate that bladder sensory input processing is dif-ferent from that of organs containing both types ofprimary afferents.

NEUROPLASTIC CHANGES INDUCED BYINTRAVESICAL VANILLOIDS ON BLADDER

SENSORY FIBERS

As seen in Figure 3, intravesical vanilloids inducea rapid and marked reduction in the number ofbladder sensory fibers immunoreactive to VR-1,substance P, or CGRP coursing in the mucosa or themuscular layer (Avelino, Cruz, Nagy, and Cruz, un-published data, 2001; and Avelino and Cruz 23).

This reduction, although �80% at 24 hours, istransient. The number of VR-1–IR fibers returns tonormal values after 1 month, and for substance-P–IR or CGRP-IR fibers after 2 months of intraves-ical vanilloid application. In contrast, galanin, apeptide usually scarcely expressed in normal sen-sory neurons, is strongly increased in DRG neu-rons after intravesical instillation of vanilloids.42

This increase lasts 1 month and is accompanied bythe overexpression of the immediate early gene c-jun for a similar period of time.42

The mechanisms underlying these neuroplasticchanges, which mimic, at least in part, those seenin sensory fibers after peripheral axotomy,43 havebeen tentatively attributed to the degeneration ofthe peripheral sensory endings in the bladder wall.

FIGURE 2. Ultrastructure of a vanilloid receptor type 1–immunoreactive fiber varicosity in the muscular layer. Thevaricosity is in close proximity to 3 smooth muscle cells, separated from them by narrow empty clefts. Small, clearand large, dense cored vesicles can be seen in the immunoreactive profile.

FIGURE 3. Mean areas of immunoreactive fibers for vanilloid receptor type 1 (solid bars), substance P (open bars),and calcitonin gene–related peptide (hatched bars) in control animals and at 24 hours, 4 weeks, and 8 weeks afterintravesical 100 nmol/L resiniferatoxin. Error bars indicate standard deviation. Asterisks indicate statistical signif-icance (*P �0.001) between mean values of control and treated animals.


However, a recent electron microscope study of ratbladders subjected to topical treatment with capsa-icin or RTX did not detect degenerated nerve fibersat 24 hours (the moment of maximal VR-1 or neu-ropeptide depletion) (Figure 4A). These are easilyidentified at electron microscopy by a shrunkencontour and a dense or dark axoplasm. However,ultrastructural changes found were restricted to apartial disruption of mitochondrial cristae and tosynaptic vesicle loss, and therefore are similar tothose previously recognized in the rabbit corneaafter topical application of capsaicin.44 Thus, suchfindings diverge from the typical ultrastructuralchanges induced by high doses of systemic capsa-icin. In this case, a rapid shrinkage and axoplasmdensification of the peripheral sensory endings oc-cur throughout all the peripheral tissues,45 includ-ing the bladder (Figure 4B).23 The strong reductionin VR-1, substance P, and CGRP immunoreactivity

should, therefore, be explained by another mecha-nism. The decrease in substance P or CGRP-IR at24 hours may be caused by the acute release ofthese peptides from peripheral sensory endingsevoked by the rapid increase of calcium inside theneurons.19 However, the prolonged effects of intra-vesical vanilloids are better explained by the down-regulation of the synthesis of substance P or CGRP,which was shown in sensory neurons after sys-temic capsaicin46 or RTX administration.47,48 Thereduced retrograde transport of neurotrophic fac-tors, in particular NGF, that are essential for thenormal metabolism of peptidergic sensory fibershas been proposed as a putative mechanism for thedownregulation of substance P25 and upregulationof galanin (Avelino and Cruz, unpublished obser-vations, 2001). Interestingly, RTX application tothe rat urinary bladder reduced in �90% the retro-grade transport of neuronal tracers from the blad-

FIGURE 4. Electron micrographs of bladder nerves coursing in the bladder mucosa 24 hours after intravesical100 nmol/L resiniferatoxin (A) or 100 mg/kg body weight subcutaneous capsaicin (B). After administration of topicalvanilloids, the large majority of terminal axons maintain a normal ultrastructure, whereas after systemic capsaicin,the axons degenerate.


der into DRG neurons, further reinforcing the roleof the axonal transport blockade to the genesis ofthe neuroplastic changes (Avelino, Diniz, andCruz, unpublished observations, 2001).

The role of the neuropeptide changes for the de-volvement of the desensitizing state is still unclear.However, it is known that substance P release inthe spinal cord facilitates reflex motor responses,49

whereas galanin has the opposite effect.50

INTRAVESICAL RESINIFERATOXIN ACTSAS AN ULTRAPOTENT, NONPUNGENT

CAPSAICIN ANALOG

Desensitization was initially believed to be pro-portional to the intensity of the pungent effect ofcompounds. This belief was, however, rapidlyabandoned after in vitro assays showed that, inequimolar doses, RTX is several thousand timesmore potent than capsaicin in inducing desensiti-zation, yet only 3 to 4 times more potent in induc-ing neurogenic inflammation or nociceptive be-havior in the eye-wiping assay.3 It was thereforeproposed that RTX could desensitize at concentra-tions so low that no significant acute irritationwould be provoked.

This hypothesis proved correct in studies thatcompared the desensitizing and pungent effects ofintravesical capsaicin or RTX by intensity of spinalc-fos gene expression in the rat spinal cord. C-fosexpression at the L6 spinal cord segment afterbladder instillation of a chemical irritant is reducedup to a complete suppression by prior bladder de-sensitization with capsaicin or RTX, the saturatingdoses being 1 mmol/L or 100 nmol/L, respective-ly.21,22 However, whereas 1 mmol/L capsaicin in-duces a strong, albeit transient, increase of the spi-nal c-fos gene 2 hours after its intravesicaladministration,51 100 nmol/L RTX induced a veryweak spinal c-fos response, comparable to that in-duced by urethral catheterization (Figure 5).22

Likewise, others have reported less nociceptive be-havior in awake rats after the bladder instillation of100 nmol/L RTX than after 100 �mol/L capsa-icin.52

The low pungency of RTX was confirmed in re-cent clinical trials.53–56 Intravesical application of50 to 100 nmol/L RTX solutions evoked phasicdetrusor contractions accompanied by urgency tourinate and a mild sensation of warmness or itch-ing in the lower abdomen.53,57 However, patientsscored the discomfort as �3 on a 10-point visualanalog scale.57 In addition, in patients with detru-sor hyperreflexia susceptible to episodes of auto-nomic dysreflexia, no cases were reported.53,54,57

These effects contrast sharply with the extremepungency of intravesical capsaicin in humans thatmanifests as an intense burning sensation requir-

ing analgesic treatment or a preliminary local an-esthesia of the bladder mucosa to be tolerable.58–60

Life-threatening episodes of autonomic dysreflexiahave also been reported after intravesical capsaicinin patients with complete spinal cord transection athigh thoracic or cervical level.61 Moreover, capsa-icin exacerbates urinary frequency, urgency, andurge incontinence during the first 2 weeks after itsapplication.58–60

The differences between capsaicin and RTX interms of pungency, binding affinity for VR-1,3,4 orcapacity for inducing calcium uptake in vitro led tothe attractive hypothesis of the existence of 2 dis-tinct vanilloid receptor subtypes13: (1) a subtypewith higher affinity for RTX and low ability forcalcium uptake was proposed to mediate desensi-tization; (2) the other, with low affinity for RTXand high capacity for capsaicin-induced calciumuptake, was proposed to mediate excitation.13

However, this concept is no longer tenable becausethe cloned rVR-1 has both high affinity for RTX inbinding assays and allows calcium currents in-duced by capsaicin.12 Thus, the different proper-ties of RTX and capsaicin might be better explainedby the existence of 2 distinct domains of the VR-1receptor with distinct binding affinities for the 2vanilloids. In this case, 1 domain is proposed to bemore effective in initiating the desensitizing path-way and the other more effective in opening thereceptor pore to extracellular ions.62

Capsaicin pungency also appears to be depen-dent on the release of nitric oxide from the centralsensory endings in the spinal cord, because noci-ceptive behavior in awake rats in response to intra-vesical capsaicin can be reduced by the intrathecaladministration of a nitric oxide synthase inhibi-tor.63 The recent identification of vanilloid recep-tors outside the nervous system may also contrib-ute to the different pungent effects of the 2vanilloids. Capsaicin, but not RTX, releases inter-leukin-4, a potent proinflammatory product, frommast cells.64

RATIONALE FOR INTRAVESICALVANILLOID APPLICATION IN DETRUSOR

INSTABILITY

In 1992, the rationale for Fowler et al.65 to initi-ate capsaicin instillations in patients with detrusorhyperreflexive of spinal origin was de Groat’s ob-servation that reflex micturition in chronic spinal-ized cats, but not in intact cats, could be sup-pressed by capsaicin.66 The explanation for thisparticular capsaicin action is the organization ofthe afferent arms of the 2 reflexes controlling mic-turition in cats. In intact animals, micturition isunder the control of a long reflex initiated in A�-fibers, whereas in chronic spinalized animals, mic-


turition is dependent on a short sacral reflex trig-gered by capsaicin-sensitive C-fibers.67 Thesuccess of capsaicin in patients with detrusor hy-perreflexia indirectly confirmed that a C-fiber–me-diated spinal micturition reflex also occurs in hu-mans.65

As in detrusor hyperreflexia, various experimen-tal and clinical observations support a potentialtherapeutic application of intravesical vanilloids inpatients with detrusor instability. In rats, detrusorinstability associated with chronic infravesical ob-struction was attributed to the emergence of ashort latency, C-fiber–mediated, spinal micturi-tion reflex.40 At least in part, this change could beattributed to the sprouting of bladder afferent ter-minals around preganglionic parasympatheticneurons lodged in the sacral spinal cord,39 possiblystimulated by neurotrophic factors synthesized inexcess in the hypertrophied bladder.38 Men withchronic bladder outlet obstruction and detrusorinstability have a high incidence of a positive ice-water test,68 suggesting a similar rearrangement ofbladder C-afferent contacts in the human spinalcord.69 In addition, in women with idiopathic de-trusor instability, the density of nerve fibers immu-noreactive for substance P or CGRP was found tobe higher than in normal age-matched women.70

Very recently, an open clinical trial of intravesi-cal RTX was initiated with patients with idiopathicdetrusor instability (Silva, Ribeiro, and Cruz, un-published observations, 2001; and Cruz and

Silva56). As in hyperreflexic patients, a single instil-lation of 50 nmol/L RTX in 12 patients with idio-pathic detrusor instability refractory to antimusca-rinic therapy caused little discomfort (averagescore �3 on 10-point visual analog scale). Whenevaluated at 3 months, 11 patients reportedmarked improvement of their urinary symptoms,particularly urge incontinence. At the 3-monthevaluation, mean (� SD) daily episodes of urinaryincontinence had decreased from 5 � 3 to 0.5 �0.6 (P �0.001), and mean daily urinary frequencyhad decreased from 12 � 3 to 10 � 3 (P �0.05). Atthe same time, mean bladder volume at the firstdetrusor contraction increased from 159 � 113 mLto 422 � 146 mL (P �0.001), and mean maximalcystometric capacity increased from 293 � 173 mLto 436 � 146 mL (P �0.05; Figure 6).

SAFETY OF INTRAVESICALRESINIFERATOXIN APPLICATION

The full acceptance of intravesical vanilloids forthe treatment of patients with detrusor overactivityis dependent on the demonstration that these com-pounds, in addition to having irrefutable therapeu-tic properties, do not induce any persistent mor-phologic changes in the bladder tissue. Capsaicinwas the first vanilloid to be used for this applica-tion, in part because it has spiced the human dietfor hundreds of years. Successive instillations ofcapsaicin in hyperreflexic patients for periods aslong as 5 years do not evoke any morphologic

FIGURE 5. Histogram showing the average number of c-fos–immunoreactive (IR) cells in L5 to S1 rat spinal cordsegments 2 hours after catheterization (solid bars), 100 nmol/L resiniferatoxin (RTX; open bars) or 1 mmol/Lcapsaicin (hatched bars). Error bars indicate standard deviation. DCM � dorsal commissure; ILG � intermediolat-eral gray matter. Asterisks indicate statistical significance (*P �0.05, **P �0.01) between mean values.


change in the bladder urothelium.71 Likewise, RTXdid not harm the human bladder in a recent patho-logic study. After repeated RTX application, blad-der cavity was normal on cystoscopic inspection,and in random biopsies, the mucosa maintained anormal morphology, including its usually thick su-perficial mucin coat.72

CONCLUSION

The identification of a specific receptor (VR-1) tovanilloids in a particular subset of sensory neuronsthat are extremely abundant in the bladder opensnew perspectives to the clinical application of in-travesical administration of those substances. Mic-turition disturbances susceptible to vanilloid treat-ment include not only detrusor hyperreflexia, butalso detrusor instability. At present, RTX is theideal vanilloid because of its low pungency andsafety. However, it is expected that new com-pounds with even better profiles will soon be re-leased by the pharmaceutical industry.

ACKNOWLEDGMENT. The author acknowledges the im-portant scientific contributions of Drs. Antonio Avelino, Car-

los Silva, and Istvan Nagy and the technical assistance of JoyceWilletts in the preparation of the manuscript.

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FIGURE 6. (A) Mean daily frequency (solid bars) and episodes of urinary incontinence (hatched bars) in 12 patientswith idiopathic detrusor instability before and 30 and 90 days after 50 nmol/L resiniferatoxin. (B) Mean bladdervolume to first contraction (solid bars) and maximal cystometric capacity (MCC; hatched bars) at the same timepoints. Error bars indicate standard deviation. Asterisks indicate statistical significance (*P �0.05, **P �0.01,***P �0.001).


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Vanilloid receptor and detrusor instability

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