Committee 2

Neurophysiology and Neuropharmacology

Chairmen

J. MORRISON (UAE),

W. D STEERS (USA),

A. BRADING (U.K.)

Members

B.BLOK (THE NETHERLANDS),

C. FRY (U.K.),

W.C DE GROAT (USA),

H. KAKIZAKI (JAPAN),

R. LEVIN (USA),

K. THOR (USA)

83

F. FUTURE RESEARCH

E. SUMMARY

IV. SUMMARY OF THE NEUROGENICMECHANISMS UNDERLYINGBLADDER OVERACTIVITY

III. GENES AND THE REGULATIONOF LOWER URINARY TRACT

FUNCTION

II. CENTRAL MODELS OF OAB

I. PERIPHERAL MODELS OF OAB

D. MODELS OF BLADDER OVERACTIVITY

III. CENTRAL PATHWAYS THATMODULATE THE MICTURITION

REFLEX

II. SUPRASPINAL PATHWAYS THATPARTICIPATE IN THE

MICTURITION REFLEX

I. SACRAL SPINAL CORD

C. CENTRAL PATHWAYS THATCONTROL MICTURITION

II. THE CONTRACTILE MACHINERY

I. EXCITATION-CONTRACTIONCOUPLING

B. SMOOTH MUSCLE

VI. PHARMACOLOGY OF PERIPHE-RAL PATHWAYS TO THE BLADDER

V. PERIPHERAL AUTONOMICINNERVATION AND GANGLIA

IV. EFFERENT NEURONS

III. SENSITIVITY OF AFFERENTENDINGS

II. AFFERENT NEURONES

I. OVERVIEW

A. PERIPHERAL NERVESTHAT INNERVATE THE

LOWER URINARY TRACT

84

CONTENTS Chapter 2

It was the view of the organising committee that the workof the two former basic science committees (Cell Biolo-gy, and Neurophysiology and Neuropharmacology)should be combined and integrated into one chapter. Thetwo chapters in the first consultation [1,2] summarisetwo perspectives on the science and biology of inconti-nence, viz (a) the central nervous mechanisms involvedin micturition and incontinence, and (b) the componenttissues of the bladder and urethra. This decision to com-bine and integrate was an important one that has causedmajor revisions and additions in an attempt to explainwhy so many components of the lower urinary tract andits innervation may change in pathological circum-stances. Consequently the subcommittee of 2001 revie-wed the neurosciences and cell biology together. Thischapter relies heavily on the report of the first consulta-tion publication [1,2], and some of this work will berepeated, sometimes in a condensed form, simply becau-se the former volume is not generally available. Thefocus of this report is predominantly on biological fac-tors that contribute to the overactive bladder, and specialconsideration is given to mechanisms that might beimportant in this state. This report also attempts to makean integrated approach, looking at all the tissues invol-ved, and the interactions between them: knowledge ofthe urothelium and its interaction with afferent nervesand the mediators involved have increased rapidly, and asection will be devoted to this topic. Properties of thebladder smooth muscle not considered in depth in theFirst Consultation, including its metabolism, contractionand relaxation, excitation contraction coupling, Ca2+

handling and electrical connectivity that are relevant tobladder overactivity are also discussed. It will be seenthat these properties affect afferent and efferent neuronesin the bladder wall, and the viability of these affectsreflex responses and sensation. Neurones affected in thisway may be found within the intramural ganglia that arelargely absent in some experimental animals, but presentin humans. New information is available on the gangliapresent in the human bladder wall and paraurethral tis-

sue, and on the interstitial cells that have been found sub-mucosally and around the smooth muscle bundles in theurethra and bladder. At the same time we will summari-se and update the previous material on neuroscience andlink it with cell biology and genetics in an attempt to pro-duce a wider view against which the plasticity of cellularcomponents of the lower urinary tract and its innervationcan be considered, particularly in relation to pathologicaldisturbances.

The overactive bladder (OAB) is a common clinical pro-blem which may originate from dysfunction of the per-ipheral or central nervous pathways, the urothelium,smooth muscle and other tissue components; in inflam-mation or cancer there may be infiltration of inflamma-tory or neoplastic cells which may also influence boththe function of nerve, muscle, urothelial and other cells.Much is known about the neural contribution to OAB,and much remains to be discovered. But there have alsobeen interesting developments in recent years on non-neural factors that might contribute to bladder hyperacti-vity, bladder instability and altered bladder sensation.Changes in the urothelium as well as the altered respon-siveness of ischaemic or denervated smooth muscleappear to be two of the most important factors in thesesyndromes. However, not only can the increased respon-siveness of the nerves be due to changes in the urothe-lium or smooth muscle, conversely changes in smoothmuscle and urothelium can be elicited by altered nerveactivity. Furthermore, long term changes in smoothmuscle (such as hypertrophy, and increased oxygenconsumption) may lead to functional ischaemia anddamage to neurones in the bladder wall. Because of theseinteractions, a vicious circle can develop in which achange in one of these components can influence theother components of the system; consequently pathophy-siological changes can be described in several compo-nents of this control system when an initial (experimen-tal) lesion has been restricted to one of these.

Models of conditions that exhibit overactive bladder andinstability have been developed, and a consideration ofthe pathophysiology of bladder outlet obstruction, blad-der inflammation, overactivity, denervation, spinal cordinjury and development or aging will be presented

INTRODUCTION

85

Neurophysiology and Neuropharmacology

J. MORRISON, W. D STEERS, A. BRADING

B.BLOK, C. FRY, W.C DE GROAT, H. KAKIZAKI, R. LEVIN, K. THOR

towards the end of the report. These models have helpedto produce an understanding of the pathophysiologicalmechanisms and are now providing a more unified hypo-thesis on the genesis of these conditions.

This chapter is therefore divided into four sections (a) theperipheral innervation (b) other peripheral tissues such asurothelium and smooth muscle, (c) the control of thelower urinary tract by the central nervous system, and (d)pathophysiological models of the overactive bladder.

This book attempts to use Levels of Evidence throu-ghout. The Oxford Centre for Evidence Based Medici-ne has laid down guidelines that apply to Levels of The-rapeutic Interventions and Grades of Recommendationsto patients; the existence of dispute regarding eachmajor conclusion should be documented. These do notreally apply to the basic sciences, where randomisedcontrolled trials are not a common format of investiga-tion, and acute studies with internal controls are morecommon.

Within this chapter we intend to be selective and reportscientific evidence that has appropriate controls andachieves statistical significance. Other categories ofevidence, e.g. uncontrolled studies, inadequate statisti-cal support, anecdotal information, hypothesis or spe-culation will be referred to as such.

Of some importance in this field are species diffe-rences, and agreement or disagreement between resultson different species will be made clear.

As indicated in Figure 1 and discussed in detail in chap-ter 1 [3], the lower urinary tract is innervated by threesets of peripheral nerves: (1) pelvic parasympatheticnerves, which arise at the sacral level of the spinal cord,excite the bladder and relax the urethra, (2) lumbarsympathetic nerves, which inhibit the bladder body andexcite the bladder base and urethra in animals, (3)pudendal nerves, which excite the external urethralsphincter and associated mechanisms in the pelvic floor[3-8]. These nerves contain afferent (sensory) axons aswell as efferent pathways.

The central pathways controlling lower urinary tractfunction are organized as simple on-off switching cir-cuits (Fig. 2) that maintain a reciprocal relationship bet-ween the urinary bladder and urethral outlet. [2, 4-9].The principal reflex components of these switching cir-cuits are listed in Table 1 and illustrated in Figure 3.Intravesical pressure measurements during bladderfilling in both humans and animals reveal low and rela-tively constant bladder pressures when bladder volumeis below the threshold for inducing voiding (Fig. 4).The accommodation of the bladder to increasingvolumes of urine is primarily a passive phenomenondependent on the intrinsic properties of the vesicalsmooth muscle and the quiesence of the parasymapthe-tic efferent pathway [4-6]. In addition in some speciesurine storage is also facilitated by sympathetic reflexesthat mediate an inhibition of bladder activity, closure ofthe bladder neck and contraction of the proximal part ofthe urethra. During bladder filling the activity of thesphincter electromyogram (EMG) also increases (Fig.4) reflecting an increase in efferent firing in the puden-dal nerve and an increase in outlet resistance whichcontributes to the maintanence of urinary continence.Motoneurones of the external urethral sphincter (EUS)are located in a region of the sacral ventral horn, calledthe nucleus of Onuf, just medial to the motoneurones ofthe hindlimb and lateral to those of the trunk and axialmusculature. In most species motoneurones of the EUSand those of the external anal sphincter are positionedin one nucleus of [10-12], but there exists a conside-rable variation among species [13-15].

The storage phase of the urinary bladder can be swit-ched to the voiding phase either involuntarily (reflexly)or voluntarily (Fig 4). The former is readily demonstra-ted in the human infant or in the anesthetized animalwhen the volume of urine exceeds the micturition thre-shold. At this point increased afferent firing from ten-sion receptors in the bladder reverses the pattern ofefferent outflow, producing firing in the sacral para-sympathetic pathways and inhibition of sympatheticand somatic pathways. The expulsion phase consists ofan initial relaxation of the urethral sphincter (Fig 4) fol-lowed in a few seconds by a contraction of the bladder,an increase in bladder pressure and the flow of urine.Relaxation of the urethral smooth muscle is mediatedby activation of a parasympathetic pathway to the ure-thra that triggers the release of nitric oxide, an inhibito-ry transmitter [16,17] and by removal of adrenergic andsomatic cholinergic excitatory inputs to the urethra.Secondary reflexes elicited by flow of urine through theurethra facilitate bladder emptying [4,5]. These reflexesrequire the integrative action of neuronal populations atvarious levels of the neuraxis. Certain reflexes such asthose mediating excitatory outflow to the sphinctersand sympathetic inhibitory outflow to the bladder are

I. OVERVIEW

A. PERIPHERAL NERVESTHAT INNERVATE THE

LOWER URINARY TRACT

LEVELS OF EVIDENCE

86

87

Table 1 : Reflexes to the Lower Urinary Tract

Afferent Pathway Efferent Pathway Central Pathway

URINE STORAGE 1. External sphincter contraction (somatic nerves) Spinal reflexesLow level vesical afferent 2. Internal sphincter contraction (sympathetic nerves)activity (pelvic nerve) 3. Detrusor inhibition (sympathetic nerves)

4. Ganglionic inhibition (sympathetic nerves)5. Sacral parasympathetic outflow inactive

MICTURITION

High level vesical afferent 1. Inhibition of external sphincter activity Spinobulbospinalactivity (pelvic nerve) 2. Inhibition of sympathetic outflow Reflexes

3. Activation of parasympathetic outflow to the bladder4. Activation of parasympathetic outflow to the urethra Spinal Reflex

Figure 1 : Diagram showing the sympathetic, parasympathetic and somatic innervation of the urogenital tract of the male cat.Sympathetic preganglionic pathways emerge from the lumbar spinal cord and pass to the sympathetic chain ganglia (SCG)and then via the inferior splanchnic nerves (ISN) to the inferior mesenteric ganglia (IMG). Preganglionic and postganglionicsympathetic axons then travel in the hypogastric nerve (HGN) to the pelvic plexus and the urogenital organs. Parasympathe-tic preganglionic axons which originate in the sacral spinal cord pass in the pelvic nerve to ganglion cells in the pelvic plexusand to distal ganglia in the organs. Sacral somatic pathways are contained in the pudendal nerve, which provides an inner-vation to the penis, the ischiocavernosus (IC), bulbocavernosus (BC) and external urethral sphincter (EUS) muscles. Thepudendal and pelvic nerves also receive postganglionic axons from the caudal sympathetic chain ganglia. These three sets ofnerves contain afferent axons from the lumbosacral dorsal root ganglia. Abbreviations: ureter (U), prostate gland (PG), vasdeferens (VD).

88

Figure 2 : Diagram illustrating the anatomy of thelower urinary tract and the switchlike function of themicturition reflex pathway. During urine storage, alow level of afferent activity activates efferent input tothe urethral sphincter. A high level of afferent activityinduced by bladder distention activates the switchingcircuit in the central nervous system (CNS), producingfiring in the efferent pathways to the bladder, inhibi-tion of the efferent outflow to the sphincter, and urineelimination.

Figure 3 : Diagram showing neural circuits controlling continence and micturition. (A) Urine storage reflexes. During thestorage of urine, distention of the bladder produces low level vesical afferent firing, which in turn stimulates (1) the sympa-thetic outflow to the bladder outlet (base and urethra) and (2) pudendal outflow to the external urethral sphincter. These res-ponses occur by spinal reflex pathways and represent Çguarding reflexesÈ, which promote continence. Sympathetic firing alsoinhibits detrusor muscle and modulates transmission in bladder ganglia. A region in the rostral pons (the pontine storage cen-ter or ÇLÈ region) increases external urethral sphincter activity. (B) Voinding reflexes. During elimination of urine, intensebladder afferent firing activates spinobulbospinal reflex pathways passing through the pontine micturition center, which sti-mulate the parasympathetic outflow to the bladder and internal sphincter smooth muscle and inhibit the sympathetic andpudendal outflow to the urethral outlet. Ascending afferent input from the spinal cord may pass through relay neurons in theperiaqueductal gray (PAG) before reaching the pontine micturition center.

organized at the spinal level (Fig 3A); whereas theparasympathetic outflow to the detrusor has a morecomplicated central organization involving spinal andspino-bulbo-spinal pathways (Fig 3B).

Normally, the micturition pathway is switched on fiveto seven times per day and this stops temporarily thetonic contraction of the pelvic floor, necessary for uri-nary continence. Neural structures responsible for mic-turition and urinary continence are located in the rostralbrainstem. They coordinate motoneurons of the urinarybladder and the EUS, both groups located in the sacralspinal cord. Interruption of the descending motor fibersfrom the pons to the sacral cord, for example in a tran-sected spinal cord, abolishes normal micturition andresults in reflex incontinence with detrusor-sphincterdyssynergia. Patients with brain lesions rostral to thepons never show detrusor-sphincter dyssynergia.However, these patients might suffer from urge inconti-nence, i.e. detrusor hyperactivity and an inability todelay voiding at an appropriate place and time. Appa-rently, centers in the pons coordinate micturition assuch, but centres rostral to the pons are responsible forthe timing of the start of micturition.

1. PROPERTIES OF BLADDER AFFERENT NEURONS.

Afferent axons in the pelvic, hypogastric and pudendalnerves transmit information from the lower urinarytract to the lumbosacral spinal cord. [6,18,19] Pelvicnerve afferents, which monitor the volume of the blad-der and the amplitude of the bladder contractionsconsist of myelinated (A-delta) and unmyelinated (C)axons (Table 2). Sensing bladder volume is of particu-lar relevance during urine storage. On the other hand,afferent discharges that occur during a bladder contrac-tion have an important reflex function and appear toreinforce the central drive that maintains the detrusorcontraction. Afferents which respond both to distensionand contraction, i.e., Ò in series tension receptorsÓ, havebeen identified in the pelvic and hypogastric nerves ofcats and rats [20-24].

In the last decade there has been an awareness of theneed to study bladder afferents under as near physiolo-gical conditions as possible to avoid sensitization ordesensitization and other effects of repeated distensionson the baseline properties of afferents [24]. Single unitstudies continue to have great value because thereappear to be different modalities of afferent fibre in thepelvic nerve, so the search stimulus for these afferents

II. AFFERENT NEURONES

89

Figure 4 : Combined cystometrograms and sphincter elec-tromyograms (EMG) comparing reflex voiding responsesin an infant (A) and in a paraplegic patient (C) with avoluntary voiding response in an adult (B). The abscissain all records represents bladder volume in milliliters andthe ordinates represent bladder pressure in cm H2O andelectrical activity of the EMG recording. On the left side ofeach trace the arrows indicate the start of a slow infusionof fluid into the bladder (bladder filling). Vertical dashedlines indicate the start of sphincter relaxation which pre-cedes by a few seconds the bladder contraction in A and B.In part B note that a voluntary cessation of voiding (stop)is associated with an initial increase in sphincter EMG fol-lowed by a reciprocal relaxation of the bladder. A resump-tion of voiding is again associated with sphincter relaxa-tion and a delayed increase in bladder pressure. On theother hand, in the paraplegic patient (C) the reciprocalrelationship between bladder and sphincter is abolished.During bladder filling, transient uninhibited bladdercontractions occur in association with sphincter activity.Further filling leads to more prolonged and simultaneouscontractions of the bladder and sphincter (bladder-sphinc-ter dyssynergia). Loss of the reciprocal relationship between bladder andsphincter in paraplegic patients interferes with bladderemptying.

has been the electrical stimulation of the pelvic nerve atvery low frequencies. This protocol avoids selectingunits by repeated mechanical stimulation of the bladder,which biases the sample towards low threshold affe-rents and can cause damage and sensitization/desensiti-zation of afferent endings, particularly when repeatedfrequently.

Afferents that respond only to bladder filling have been

identified in the rat bladder, and appear to be volumereceptors, possibly sensitive to stretch of the mucosa. Inthe cat bladder some Òin series Ò tension receptors mayalso respond to bladder stretch [25]. A few afferents thatcan be activated at high bladder volumes or pressureshave been identified in the rat and the cat [21,23,24,26,27]. It is unclear to what extent the demonstration ofthese afferents depends upon sensitization induced byrepeated high pressure (80 mm Hg) bladder distensions[26], which may have induced a pathological change inthe bladder and affected the properties of some of theafferents that were studied later in the experiments.

Species differences as well as differences of nomencla-ture might account for some of the variations in repor-ted properties of bladder afferents. For example, theconduction velocity which differentiates A-delta and C-fibres in the cat is 2 meters/sec, whereas in the rat it is1.3 meters/sec[28]. In the cat, A-delta bladder afferentsappear to be low threshold mechanoreceptors [22],whereas some C-bladder afferents [29] are mechano-insensitive (Òsilent C-fibersÓ) (Table 2). Other ÔsilentÕafferents in the cat may be nociceptive, and have beenfound to be sensitized by intravesical administration ofchemicals [29]. However in the rat there is now evi-dence that a significant proportion of C fibres are acti-ve at physiological volumes , i.e. the proportion ofÔsilentÕ afferents is less than had been previously esti-mated [24,30], and the link between C fibres and noci-ception becomes more complex than previously reco-gnised, because the ÔsilentÕ group of fibres includessome A-delta as well as some C fibres afferents. In a

recent well-controlled study [31] it was found that C-fibres afferents in the rat are often mechanosensitiverather than nociceptive, and that some A-delta fibres areÔsilentÕ afferents. Some of the afferents with highmechanical thresholds were not in the body of the blad-der but at the vesico-ureteric junction; the location andthe different elastic properties at this site can explainsome of the differences in the functional properties.Some of the ÔsilentÕ afferents responded to capsaicin orpotassium and were regarded as chemoreceptors.However, in the rat there is now evidence that C-blad-der afferents behave as volume receptors and that themajority do not respond to bladder contractions. Sur-prisingly, in comparison to the cat, one third of the C-afferents tested behaved as Òin series tension recep-torsÓ, which respond to both distensions and contrac-tions [24, 30]. There is also evidence that hormonal sta-tus can change the properies of bladder afferents [31].Thus the division between physiological, nociceptiveand ÔsilentÕ afferents is not sharp, and it would be unwi-se to infer function simply on the basis of conductionvelocity.

2. AXON COLLATERALS AND AXON REFLEXES

The peripheral axons of most afferent fibres divide intoa series of axon collaterals in the organs they innervate,and these axons can release neurotransmitters on tovarious tissues, including vessels, smooth muscle, uro-thelium and other neurones. These axon collateralsmediate axonal reflexes, which are the basis for neuro-genic extravasation and inflammation [32]. There isevidence in the human bladder that intramural neuronesreceive axonal contacts that contain the peptides cha-racteristic of primary afferents (see the section on blad-der ganglia and smooth muscle). Afferent and efferentfibres have close connections with mast cells in themucosa and ganglia of the guinea-pig bladder [33] andultrastructual evidence suggested that the communica-tion between the cells might be bi-directional. It wassuggested that these structures participate in axon

90

Table 2 : Properties of bladder afferents

Fiber-Type Location Normal Function Effects of Inflammatory Mediators

A-DELTA (finely smooth muscle Respond to muscle Increased discharge during distensionmyelinated axons) wall tension and lowered pressure thresholds

C-FIBER mucosa Respond to stretch of Increased discharge during distension(unmyelinated axons) mucosa (Bladder Volume Sensors) and lowered pressure thresholds

C-FIBER mucosa Insensitive to normal bladder Afferents become mechanosensitive(unmyelinated axons) distension (ÇSilentÈ Afferents) thereby unmasking a new afferent

pathway during inflammation

C-FIBER ?mucosa Sensitive to overdistension ? Sensitive to some irritants(unmyelinated axons) ?muscle (Nociceptors)

?serosa

reflexes that regulate normal vascular and detrusorsmooth muscle function and cause vasodilatation, oede-ma, inflammation, and bladder hyperactivity. Otherpeptides that can be released by axonal reflexes includeCGRP, a breakdown product of which is known to havechemotactic properties [34]. Further evidence for theinvolvement of mast cells in interstitial cystitis and theparticipation of NK-1 receptors in antigen induced cys-titis indicated a mandatory participation of these recep-tors in the chain of events linking mast cell degranula-tion and inflammation [35]. In normal mice, an antigenchallenge causes activation of mast cells, plasma extra-vasation and migration of neutrophils; however none ofthis occurs in the NK-1 receptor knockout mouse,which appears to be protected from inflammation indu-ced by an antigen challenge.

3. SPINAL PROJECTIONS OF LOWER URINARY

TRACT PRIMARY AFFERENT NEURONS

Axonal tracing experiments [18,36,37,38] have locali-zed the segmental distribution and spinal termination ofafferent pathways in the pelvic, hypogastric and puden-dal nerves. The primary afferent neurons of the pelvicand pudendal nerves are contained in sacral dorsal rootganglia; whereas afferent innervation in the hypogastricarises in the rostral lumbar dorsal root ganglia. The cen-tral axons of the dorsal root ganglion neurons carry thesensory information from the lower urinary tract tosecond order neurons in the spinal cord. Transganglio-nic transport of axonal tracers has identified the spinalprojections and terminal fields of visceral and somaticprimary afferent neurons (Fig 5).

Visceral afferent fibers of the pelvic [37] and pudendal[38] nerves enter the cord and travel rostrocaudallywithin LissauerÕs tract. Axon collaterals from LissauerÕstract distribute transversely around the lateral and medialedges of the dorsal horn (Figures 5 and 6). These distri-butions termed the lateral (LCP) and medial collateralpathways (MCP) of LissauerÕs tract, respectively, carryaxons to deeper layers of the spinal cord. Within the spi-nal gray matter, the LCP and MCP provide a dense inner-vation to laminae I, V, and VII and the dorsal commissu-re. Muscle and cutaneous afferents in the pudendal nerveterminate in different regions of the cord. Central projec-tions of pudendal nerve afferents from the external ure-thral sphincter muscle have been shown to overlap withthose of visceral afferents in the pelvic nerve. However,pudendal nerve cutaneous afferents innervate a terminalfield within the medial third of the dorsal horn in laminaeIII and IV. Proprioceptive afferent fibers from the levatorani muscle are distributed most densely in medial laminaVI. This region is known to contain second order inter-neurons that mediate reflexes initiated by Golgi tendonorgans and muscle spindles.

4. NEUROTRANSMITTERS

Visceral afferent neurons contain a number of peptider-gic neurotransmitters, and the central distribution ofbladder afferent terminals and peptidergic immunoreac-tive fibers is quite similar [18,39] This similarityincludes the periodic nature of their distribution in theLCP and their transverse projections through the inter-mediate gray matter. It has recently been shown thatchanges in the peptidergic nerves to the bladder occurin chemically induced cystitis [40,41]. Increasedexpression of CGRP, Substance P and pituitary adeny-late cyclase-activating polypeptide (PACAP), a relativeof VIP, are present in the spinal projections of pelvicnerve afferents following cyclophosphamide inducedcystitis. Within the spinal cord, PACAP appears to faci-litate micturition when administered intrathecally, andalso has excitatory actions on the spinal slice prepara-tion [42]. Electrophysiologic studies on spinal cordslices from neonatatal rats reveal that this transmitteracts on interneurons that project on to preganglionicneurons [43]. Nonpeptide neurotransmitters, such asglutamic acid [44,45] and ATP also play a role in affe-rent pathways from the urinary tract.

5. UROTHELIUM : AN ACTIVE MEMBRANE

The urothelium is much more than the classical barrierthat separates urine from the extracellular fluid. It isalso an active absorptive epithelium which absorbssodium, using amiloride sensitive sodium channels inthe urothelial cell membrane. The number of thesechannels is controlled by the level of cAMP in the cell[46], and by the sodium balance of the animal [47]. Theurothelium is also a secretory tissue that secretes urina-ry proteins such as tissue plasminogen activator andurokinase as well as a protease inhibitor using a Ca2+

regulated pathway [48].

The possibility that there may be some active controlover the functional state of the urothelium has beenrecognised for some time. A variety of adrenergic andpeptidergic receptors are present in the urothelium ofthe rat and other species. In the rat, b-1 receptors werepredominant in the bladder neck, and b-2 receptors inthe bladder dome. In addition, surgical denervation cau-sed an increase in b-1 receptors throughout the wholebladder, which suggested that there was some functio-nal regulation of receptor expression in response todenervation [49-51]. Neurokinin receptors (NK-1, NK-2 and NK-3 receptors) were also found in the mucosaas well as CGRP receptors in the urothelium of thebladder and urethra [50,51]. More recently the VR1receptor which is activated by capsaicin and protonshas also been found in the basal and superficial cells ofrat urothelium and in nerve fibres that penetrate the uro-thelium [52]; the latter staining was reduced following

91

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Figure 5 : Comparison of the distribution of bladder afferent projections to the L6 spinal cord of the rat (A) with the distri-bution of c-fos positive cells in the L6 spinal segment following chemical irritation of the lower urinary tract of the rat (B) andthe distribution of interneurons in the L6 spinal cord labeled by transneuronal transport of pseudorabies virus injected intothe urinary bladder (C) Afferents labelled by WGA-HRP injected into the urinary bladder. C-fos immunoreactivity is presentin the nuclei of cells. DH, dorsal horn; SPN, sacral parasympathetic nucleus; CC central canal. (D) Diagram showing thelaminar organization of the spinal cord.

Figure 6 : Neuroanatomical distribution of primary afferent and efferent components of storage and micturition reflexeswithin the sacral spinal cord. For purposes of clarity, afferent components are shown only on the left, while efferent compo-nents are shown only on the right. Both components are, of course, distributed bilaterally and thus overlap extensively. Visce-ral afferent components (pink and green regions) represent bladder, urethral, and genital (glans penis/clitoris) afferent fiberscontained in the pelvic and pudendal nerves. Cutaneous perineal afferent componets represent afferent fibers that innervatethe perineal skin contained in the pudendal nerve. Muscle spindle afferent components represent Ia/b afferent fibers contai-ned in the levator ani nerve that innervate muscle spindles in the levator ani muscle.SPN sacral parasympathetic nucleus LCP lateral collateral projection MCP medial collateral projection

treatment with capsaicin, which suggests that theseVR1 receptors were on afferent nerve endings. TheVR1 receptor has also been identified in human urothe-lium [589]. In urothelial cells cultured from cat blad-ders, functional evidence of P2Y receptors was found innormal animals, and of P2X receptors in cats with feli-ne interstitial cystitis [53]. All in all this indicates thatthe urothelial cells have a number of important recep-tors on their surface: adrenoceptors, CGRP receptors,VR1 receptors as well as neurokinin and purinergicreceptor subtypes.

So there is a firm basis to the conclusion that the uro-thelium may be a target for the action of certain neuro-transmitters including amines, purines and peptides.The responses of this tissue to such stimuli include theproduction of mediators that may act within the bladderwall.

6. MUCOSAL AFFERENTS

There is now good ultrastructural evidence that there isan afferent innervation of the epithelium whichinvolves not only afferent endings underneath the lami-na propria, but also afferent nerve endings within theurothelium itself. Intramural axons have been identifiedin frozen sections and in whole mount preparations ofrat bladder mucosa and smooth muscle using synapto-physin-immunofluorescence and CGRP immunoreacti-vity as a marker for afferent axons [54]. These afferentaxons were distributed over four distinct targets: (a) thebase of the epithelium, (b) inside the epithelium, (c) theblood vessels (both arteries and veins) and (d) themuscle bundles. In the mucosa, all the afferent axons,except the perivascular ones, lay either inside the epi-thelium itself or in a subepithelial plexus very close tothe basal surface of the epithelium, where multiplebranching was common. The plexus was thickest in theneck of the bladder and in the initial portion of the ure-thra, and it became progressively less dense in the adja-cent regions. It did not extend beyond the equatorialregion, and therefore the mucosa of the cranial regionof the bladder had no afferent axons. In contrast, theafferent innervation of the musculature was more diffu-se, and appeared uniform throughout the bladder.CGRP-immunofluorescence in mucosal afferent axonswas enhanced in the surviving axons 5 days aftercontralateral denervation, a change which may be anearly sign of regeneration of these axons. In the humanbladder, CGRP together with Substance P and NKAoccur only infrequently in nerves in the muscle but aremoderately frequent in the suburothelial layer. Also inthe human there appears to be another population ofCGRP-containing fibres that co-localize with NPY andgalanin and some of these synapse on intramural gan-glia within the bladder [55-58]. There is also recent

evidence that nerve endings are found crossing thebasal lamina and entering the basal layers of the humanurothelium [59]. Substantial cross over of afferentsfrom one side of the bladder to the other was presentand was also found in tracing studies [60]. Neverthe-less, the former authors did observe that unilateraldenervation did leave some areas of the bladder wallwithout afferent innervation.

Levin et al [61] found that removal of the urotheliumsignificantly and substantially increased the contractileresponse of the cat bladder to to electrical field stimula-tion, and to potassium, bethanechol and phenylephrine.They concluded that in this species, the mucosa has asignificant inhibitory effect on the contractile response ofthe bladder to stimulation. There are a series of media-tors, including nitric oxide and ATP that may be releasedby the mucosa, and act on the urothelium, the afferentand efferent nerves, and on the smooth muscle itself.

1. ROLE OF ATP AND P2X3 RECEPTORS

Recent studies of mice have shown that one of the ATPreceptors, the P2X3 receptor, is present in small sensoryneurones innervating the bladder mucosa, and that theeffects of bladder distension on these sensory endings ismarkedly attenuated if the gene for the P2X3 receptor isdeleted. P2X3 knockout mice show major changes insensation and in the function of the lower urinary tract,so severe that the bladder capacity is increased, the cys-tometrograms indicate the presence of hyporeflexia, andthe frequency of urination is reduced. This receptor isone of many purinergic receptors present in the bladderand lower urinary tract, and the topic is sufficientlyimportant to summarise the salient features of the P2Xfamily of receptors and their distribution in rodents andhumans. Of these, at least 7 subtypes of P2X receptorsappear in the rat bladder [62]. Their distribution isshown in the following table, which indicates that ATPmust have a major role to play within the urothelium,smooth muscle and neurones of the bladder wall.

The P2X3 receptor is of particular interest with respectto the physiology of pelvic nerve afferents because it isselectively expressed on small diameter sensory neu-rons, and knockout mice that do not express this recep-tor exhibit a marked urinary bladder hyporeflexia, cha-racterized by decreased voiding frequency and increa-sed bladder capacity, but normal bladder pressures[63,64]. In addition, these null mice have reduced pain-related behaviour in response to injection of ATP andformalin; and they lose the rapidly desensitizing ATP-

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induced currents in dorsal root ganglion neurons andhave a reduction in the sustained ATP-induced currentsin nodose ganglion neurons. Immunohistochemical stu-dies localize P2X3 to nerve fibres innervating the uri-nary bladder of wild-type mice, and show that loss ofP2X3 does not alter sensory neuron innervation densi-ty. Thus, P2X3 is critical for peripheral pain responsesand afferent pathways controlling urinary bladder volu-me reflexes, which take place at physiological volumesand pressures. Antagonists to P2X3 may therefore havetherapeutic potential in the treatment of disorders ofurine storage and voiding such as overactive bladder.

The idea that ATP is released from urothelial cells byexposure to raised hydrostatic pressure was proposedby Ferguson et al [65] and this idea was followed up inin vitro experiments in the rat by Navasivayam et al[66]. During normal cystometry the number ofimpulses generated in the afferent neurons was halvedafter treatment with the P2X antagonist suramin, whichindicated that a substantial part of the responses couldbe generated by the chemical purinergic signal ratherthan by a mechanosensitive mechanism. Suraminhowever is not a specific antagonist, but confirmationof the main conclusion (that mechanosensitivy is atleast partly mediated by sensitivity to ATP released bystretch of the tissues) came from recent studies [67] ofknockout mice, which indicated that ATP was releasedin the mucosa during bladder distensions, and that thisrelease was also present and unchanged in the knockoutmice. The afferent response to distension however wasgreatly attenuated in the knockout animals, indicatingthat the mechanosensitive properties of these afferentsare probably largely associated with their sensitivity toATP released within the bladder wall. Most recently,there has been a single unit analysis of bladder afferentsfrom wild and knockout mice that also used a muchmore specific P2X3 receptor antagonist [68]. These stu-dies confirmed that in this species much of the mecha-noceptive response of the afferents was mediated byATP. It appears therefore that the afferent limb of themicturition reflex is mediated to a large extent by che-moreceptors that respond to the local release of ATP

during distensions. Furthermore, this chemosensitivityis active in the normal range of volume and pressure,and suggests that the afferent pathways of the micuri-tion reflex are not confined to endings in the smoothmuscle layer, and that stretch of the urothelium is alsoinvolved. The P2X3 and P2X5 receptors on the smoothmuscle at the site of contacts with parasympathetic neu-rones are lacking in patients with detrusor instabilityand urge [69]; some further discussion of this is contai-ned in the section on efferent innervation of the bladder.(Fig. 7)

2. ROLE OF NITRIC OXIDE

Nitric oxide is an important mediator that can be relea-sed from urothelium and from neurones in the submu-cosal plexus. The detrusor however is not very sensiti-ve to nitric oxide in contrast to the outflow regionwhere it effectively relaxed the smooth muscle, which

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Table 3 : Purinergic receptors

Subtype Distribution of Puringeric Receptor Subtypes in Rodent Bladder [62,63,76,151]

P2X1 the main purinergic receptor present in the smooth muscle cell membranes.

P2X2 present within smooth muscle

P2X3 present in nerve bundles within the detrusor and in submucosal afferents

P2X4 present in capillaries in the detrusor and lamina propria,

P2X5 a strong reaction from urothelial cell membranes

P2X6 in the basement membrane below the urothelium

P2X7 present in urothelial nuclei

Figure 7 : This diagram of the relationship between theurothelium and an afferent nerve endings indicates thesources and effects of two important mediators Ð ATP andnitric oxide. The P2X3 receptor is a purinerguic receptor onafferent nerve endings that is believed to be intimately invol-ved with the process of mechano-transduction. Noradrena-line (NA) is co-released along with ATP from sympatheticnerve endings. Nitric Oxide (NO) acts on guanylate cyclase(GC) in the afferent nerve endings.

suggests an involvement of nitric oxide in the decreasein urethral pressure at the start of micturition [70].

NO may be involved in the control of afferent sensiti-vity, and we now know that NO may in some circum-stances, such as spinal cord injury, increase the activityof capsaicin-sensitive nerves within the bladder wall[71]. Basal release of nitric oxide has not been detectedin the urothelium of the normal cat; however it is relea-sed in cats with feline interstitial cystitis [53], and fromnormal cats after the addition of agonists. Nitric oxiderelease from neurones and from the urothelium hasbeen studied also in a variety of pathophysiologicalmodels using direct measurements. In one model ofinterstitial cystitis created by injecting DMSO into therat bladder, there was an increase in reflex activity inpelvic efferent neurones, a decreased bladder capacityand increased afferent activity in capsaicin sensitivebladder afferents (as indicated by the development of c-fos expression in sensory pathways). In that study [72],NO was released from dorsal root ganglion cells andfrom urinary bladder strips by the addition of DMSO orcapsaicin. In addition in normal bladders it was shown[73] that capsaicin causes NO release from the urothe-lium and from the bladder nerves, while norepinephri-ne caused NO production and release from the urothe-lium only. Norepinephrine of course can be releasedfrom sympathetic nerve terminals along with ATP andNPY, and this could be one mechanism by which thebladder is affected in spinal cord injury. Increasedexpression of neuronal NOS in bladder afferent and spi-nal neurones occurs following cord injury [74], and inbladder afferents following chronic bladder irritationwith cyclophosphamide. Nitric oxide can act on cul-tures of urothelial cells to lower paracellular epithelialresistance [73], and this damaging effect on the urothe-lium may allow other mediators to act on sensoryendings. Interestingly, the deleterious effects of nitricoxide on urothelial resistance can be blocked with GM1ganglioside. There is also evidence that nitric oxide caninhibit the function of primary afferent neurons [590,591].

Nitric oxide can therefore be produced by the urothe-lium itself and by some of the neurones in the bladderwall and the vessels. This released NO can act on anumber of targets, including sensory neurones and theurothelium itself. Knockout mice that do not have neu-ronal NOS appear to have normal function in the lowerurinary tract ([75], and knockout animals that do nothave inducible NOS are not particularly abnormal.However, the latter appear to need iNOS in the respon-se to urinary obstruction [76]. So there is clear eviden-ce that in certain models nitric oxide is an importantmediator, but possibly not so much in normal animals.

3. ROLE OF TACHYKININS AND NK-2 RECEP-TORS

An electrophysiological study of pelvic nerve afferentsduring slow cystometry in the rat also found evidenceof three types of response that characterised threeclasses of afferent neurones [31,77,78]. Some afferentswere silent, and did not respond to distension of thebladder; most of these were C-fibres. Of those that didrespond to distension, one subgroup gave the classicalÔin seriesÕ type of response characterised by excitationduring distension and during bladder contraction; mostof these were A-delta fibres, but there was also a signi-ficant contribution from C-afferents. However, manyafferents (including A-delta and C-fibres) respondedonly to distension, and it seems likely that these werepresent in the mucosa if not in the epithelium. Thesemay be regarded as volume receptors or mucosal recep-tors, and significantly with respect to the latter location,they also respond to chemicals introduced within thebladder itself. These properties rather favour the viewthat these endings exist in the submucosa. They are pro-bably best described as polymodal in their responses,and their excitability can be increased by a number ofintravesical chemical stimuli. These nerve endings pro-bably correspond with those described histochemicallythat contain CGRP, Substance P and NKA, and some ofthese afferents are also capsaicin-sensitive. They proba-bly take part in axon reflexes within the bladder muco-

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Figure 8 : This diagram of the relationship between theurothelium and an afferent nerve endings indicates thesources and effects of other important mediators Ð thevanilloid receptor (VR-1) ,which influences nitric oxide andATP release, and nerve growth facto (NGF).

sa and release mediators including CGRP, Substance Pand Neurokinin A, and are probably the source of thetachykinins that act on the NK-1 and NK-2 receptors onmast cells, smooth muscle and other nerve endings.

The view that tachykinins can sensitize sensory nerveendings is based (a) autoradiographic studies that showthe disappearance of NK-2 receptors in the submucosain capsaicin-treated rats that are deficient in sensorynerves [51], (b) on studies in which afferents can bemade hypersensitive using a NKA- analogue and otherintravesical chemical stimuli such as high [K+] andhigh osmolality, and (c) the demonstration that thedevelopment of hypersensitivity to a number of sensiti-zing agents including high [K+] can be blocked by anNK-2 receptor antagonist [31,77]. More recently it hasbeen shown that rat dorsal root ganglion neurones areexcited by NK2 agonists, but are inhibited by NK-3agonists [79]. This NK2 action is on L- and N-typeCa2+ channels, whereas the NK-3 action is only on theL-type channels. Both of these effects are blocked byinhibition of protein kinase C.

4. ROLE OF VANILLOID (VR-1) AND OPIATE

RECEPTOR-LIKE (ORL1) RECEPTORS

Mention has already been made of the actions of cap-saicin on the urothelium and nerves. The release of NOand the increase in intracellular Ca2+ induced by cap-saicin are blocked by the VR-1 antagonist capsazepine.The actions of capsaicin can also be facilitated by pro-tons, which is of interest because the VR-1 receptor isknown to be sensitive to protons. Whenever a membra-ne receptor is the site of action of an exogenous agent(for example, morphine acting on opiate receptors), thepossibility of an endogenous agent (enkephalins, in thecase of opiate receptors) indicates the possibility thatthere are new endogenous compounds that may play arole in physiology or pathophysiology. Anandamideand Nociceptin are two such compounds and are suffi-ciently new to deserve a mention, although much morework needs to be done to elucidate their roles.

a) Anandamide is an endogenous cannabinoid whichalso is an agonist of VR-1 receptors [80,81] and acts onperipheral perivascular sensory terminals in a mannerthat is antagonised by the capsaicin antagonist capseze-pine. It can cause the release of CGRP and Substance Pby increasing intracellular Ca2+, and has other actions,such as activation of G-proteins [82]. The role of anan-damide in the lower urinary tract is unknown, but theidentification of a naturally occurring agonist of theVR-1 receptor is worth noting. The VR-1 (capsaicin)receptor is a cation channel expressed by nociceptiveneurones and can also be activated by protons or tem-perature greater than 43 degrees C [83,84]. Within thebladder, it may be that it is activated naturally by low

pH, but such changes (e.g. in metabolic acidosis) arenot usually associated with bladder pain. The expres-sion of the VR-1 receptor in sensory neurones is regu-lated by Nerve Growth Factor (NGF), and concomitantwith its expression, stimulation of the VR-1 receptorwith capsaicin causes the release of CGRP [85].

b) Nociceptin, another endogenous ligand that bindswith the opioid receptor-like 1 receptor (ORL1 recep-tor) has been shown to have naloxone resistant inhibi-tory effects on the micturition reflex. These actions aremediated at several sites including the capsaicin sensiti-ve nerves in the bladder, and a central supraspinal site[86]. Nociceptin (100 nmol/kg) produced a long-lastingprotection from capsaicin desensitization of the afferentnerves that mediate the chemoceptive micturition reflex.In fact a chemoceptive micturition reflex could berepeatedly evoked by topical capsaicin in nociceptin-pretreated rats. This is in sharp contrast to the effects ofnociceptin on the local response to capsaicin which cor-respond to the ÔefferentÕ function of capsaicin-sensitiveafferent neurons. Taken together, these results suggestthat the afferent and ÔefferentÕ functions of capsaicin-sensitive primary afferent neurons in the rat bladder aredifferentiated by nociceptin, and that nociceptin has asignificant action on afferent sensitivity.

Local adminsitration of kappa-opioid receptor agonistsby intra-arterial injection attenuated the responses ofpelvic nerve afferents from the bladder to high pres-sures distension of the urinary bladder [87]. These ago-nists had essentially the same effects whether the blad-der was inflammed or not. The conclusion was that theability of kappa opioid agonists to attenuate the res-ponses of afferents to large bladder distensions indica-ted a potential use for peripherally acting kappa opioidreceptor agonists in the control of urinary bladder pain.

5. ROLE OF NEUROTROPHINS

Nerve Growth Factor (NGF; neurotrophin-1), the firstof a group of growth factors called neurotrophins, isproduced in larger quantities in humans with detrusoroveractivity [88], interstitial cystitis and bladder cancer[89], in rats with inflammed bladders [90], spinal cordinjury or chemically induced cystitis [40] or bladderoutlet obstruction [91], in diabetic rats [92] and a num-ber of other states. This protein is known to sensitizeafferents from the bladder [93] and it is involved in theproduction of referred pain in bladder inflammation[94]. It also appears to stimulate the expression of thevanilloid receptor VR-1 [85] (Figure 9). Dmitrieva andMcMahon (1996) demonstrated a role for NGF in sen-sitizing bladder afferents [95]. After filling the bladderwith human recombinant NGF, the large majority ofafferents, both myelinated and unmyelinated, becamesensitised and some of the initially non-mechanosensi-

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tive became mechanosensitive. The sensitisation beganwithin 30 min of exposure to NGF, and persisted for atleast 3 hours. NGF also caused a dose-dependent extra-vasation of EvanÕs blue into the bladder, which suggeststhat axonal reflex mechanisms may be activated. Admi-nistration of NGF into the bladder causes bladderhyper-reactivity in Wistar, but not in Sprague-Dawleyrats [96], and the effect was not influenced by pretreat-ment with capsaicin. This report concluded that the A-delta afferents were involved particularly in the deve-lopment of hyper-reactivity. There is also evidence ofreferred pain following instillation of NGF into thebladder [85].

Clemow et al (2000) found that cyclic and static stret-ching of bladder smooth muscle cells stimulates increa-sed NGF production. This was particularly pronouncedin certain strains of rats (e.g. the spontaneously hyper-tensive rat and the Wistar-Kyoto Hypertensive rat).Another source of NGF is the vascular smooth muscle,and gene transcription, intracellular Ca2+, protein kina-se C (PKC), and autocrine release of an unknown factorappear to play a part in secretion from vascular andbladder smooth muscle. Basal cytosolic Ca2+ appears tobe particularly involved in the regulation of NGF secre-tion in bladder and vascular smooth muscle cells [97].

Recently NGF gene insertion into the bladder and blad-

der afferent pathways as a possible treatment for diabe-tic cystopathy in rats, using herpes simplex virus type 1as a vector, has been investigated [98]. The expectationwas that the NGF gene may be transferred to the affe-rent neurones using the virus and that the expression ofNGF would be increased, thus improving the availabi-lity and transport of NGF within neurones that are defi-cient in the protein. This experiment was successful andclearly points to one way forward in the potential treat-ments of this condition.

Brain Derived Neurotrophic Factor (BDNF) levels inthe urinary bladder and some other epithelia are higherthan those found in the brain or skin [99]. These authorsconcluded that visceral epithelia are a major source, butnot a target, of BDNF in the adult viscera. The abun-dance of BDNF protein in certain internal organs sug-gests that this neurotrophin may regulate the function ofadult visceral sensory and motor neurons. In situ hybri-dization experiments showed that BDNF mRNA wasmade by visceral epithelial cells, in several types ofsmooth muscle, and in neurons of the myenteric plexus.However the target receptors (trkB and p75[NTR])were not present on the urothelium but were present inon neurons of the peripheral nervous system. Hence theneurotrophin is produced by the urothelium and can acton the nerves.

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Figure 9 : This diagram summarises the relationships between different tissue component and the afferent nerve endings,and the role of different mediators.

The mRNAs for NGF, BDNF and neurotrophin-3 allincrease within 2 hours of bladder inflammation in therat, and these were responsible for mediating thechanges in sensory and reflex hyperactivity. Howeverthe increases in neurotrophic factor concentrations arenot maintained despite an increase in their mRNAs[40,90]. These changes were most marked 4 days afteracute spinal cord injury when the mRNAs for b-NGF,BDNF, Glial-Derived Growth factor [GDNF], CiliaryNeurotrophic factor [CNTF], and neurotrophins 3 and 4[ NT-3 and NT-4 ] were all grossly elevated. It was alsoconcluded that these mRNAs change during develop-ment and that there is a relatively high expression ofNT-3 and NT-4 protein in the adult urinary bladder,which suggests a potential importance of these factorsin the adult lower urinary tract [40].

An approach to the problem of neuroprotection hasbeen followed recently using antigen specific T-cellsthat target areas of damaged nerve and release neuro-trophins at those sites so as to provide local neuropro-tection. This mechanism is given the term neuroprotec-tive autoimmunity [100]. In intersitital cystitis it isknown that different types of inflammatory cells infil-trate the bladder wall [101,102]. These include T-cells,B-cells and macrophages, and interest has recentlyfocused on the role of these cells in the type of inflam-mation found in this condition. Christmas [102] sug-gested that the increased numbers of CD4+, CD8+ andgamma delta cells as well as IgA+, IgG+ and IgM+plasma cells within the urothelium and submucosa inpatients with IC play an active role in the pathogenesisof the disorder. More recently there is evidence thatneurotrophins can be released from autoimmune T-cellsthat are reactivated on meeting a specific antigen: thelist of neurotrophins that can be released includes NGF,BDNF, NT-3 and NT-4/5. The mRNAs for the neuro-trophin receptors TrkA, TrkB and p75 can be expressedin injured nerves, and it has been suggested that thesespecific receptors can mediate the effects of T-cell deri-ved neurotrophins [103]. They also presented evidencethat the neurotrophins were mediators that providedneuroprotection and suggested that T-cell interventionin the injured CNS may be a useful means of promotingprotection by the supply of neurotrophins. The idea ofneuroprotective autoimmunity is relatively recent in theliterature [100] but is seen as a potential therapy in anumber of disorders involving nerve injury. It is doubt-ful if T-cell mediated mechanisms are involved in mostof the acute injury models studied in the lower urinarytract, but it seems quite possible that such mechanismsmay be involved in chronic states such as interstitialcystitis. Recently a Japanese study using a new immu-noregulator (IPD-1151T ) that suppresses helper T cellmediated allergic responses has shown an improvementin symptoms and a reduction in inflammatory cellpopulations [104].

6. EFFECTS OF REPETITIVE NERVE STIMULA-TION ON BLADDER ACTIVITY; INFLUENCE OF

URETHRAL AFFERENTS

The functional properties of reflexes from the rat andcat bladder can be influenced by repetitive electrical sti-mulation, which causes an increased excitability of themicturition reflex and a lowering of micturition thre-shold. It has been known for many years that repetitivestimulation of A-delta afferents can alter the excitabili-ty of the micturition reflex in the cat [107]. It hasrecently been suggested that the prolonged enhance-ment of excitatory synaptic transmission is due to faci-litation of the central micturition reflex pathway[105,106,108]. The same group also provided evidenceof facilitation of the micturition reflex from stimulationof uretheral afferents, and of inhibition of bladder acti-vity by stimulation of the dorsal nerve of the clitoris[108], which is in keeping with known interactionsfrom the vagina and colon [4,109]. Stimulation of ure-thral afferents by flowing fluid through the urethra canfacilitate the micturition reflex; however contraction ofthe urethral sphincter resulted in inhibition of bladdermotility [110].

1. PARASYMPATHETIC PREGANGLIONIC

NEURONS

Bladder parasympathetic preganglionic neurons (PGN)are located in the lateral part of the sacral intermediategray matter in a region termed the sacral parasympa-thetic nucleus (SPN) (Old Fig. 6). The PGN are small ,fusiform-shaped cells which send dendrites into laterallamina I of the dorsal horn , the lateral funiculus andmedially into the dorsal commissure. The dendritricstructure very likely indicates the origin of importantsynaptic inputs to the cells.[5,35,36,37,114,115]. Blad-der PGN send axons through the ventral roots to per-ipheral ganglia where they release the excitatory trans-mitter, acetylcholine[111]. In some species they releaseopioid peptide transmitters and express nitric oxidesynthase [112], suggesting that they also release nitricoxide. The reflex activity and control of the parasym-pathetic neurones innervating the bladder is the subjectof the section on Central Nervous Control.

2. EXTERNAL URETHRAL SPHINCTER

MOTONEURONS

External urethral sphincter (EUS) motoneurons arelocated in a circumscribed region of the sacral ventralhorn, OnufÕs nucleus (Fig. 6). In primates and carni-vores, OnufÕs nucleus is located along the lateral border

IV. EFFERENT NEURONS

98

of the ventral horn [38]. Within the nucleus, urethralmotor neurons occupy a ventrolateral position and analmotor neurons occupy a dorsomedial position. Sphinc-ter motoneurons exhibit tightly-bundled dendrites thatrun rostrocaudally within the confines of the nucleus. Inaddition to the rostrocaudal dendritic projections,sphincter motor neurons also exhibit transversely orien-ted dendritic bundles that project laterally into the late-ral funiculus, dorsally into the intermediate gray matter,and dorsomedially toward the central canal. This den-dritic pattern is similar to that of bladder PGN and verydifferent from that of limb motoneurons, suggestingthat EUS motoneurons and PGN receive inputs fromsimilar regions of the spinal cord. EUS motoneuronssend axons through the ventral roots and into thepudendal nerves.

¥ Pudendal nerve reflexes

The activation of urethral striated muscle sphincter neu-rons in response to stimulation of bladder (pelvic nerve)or urethral/perineal (pudendal nerve) afferents is part ofa continence-maintaining mechanism. These reflexesrecorded as efferent discharges on the pudendal nervein chloralose-anesthetized cats were suppressed by thea1 adrenoceptor antagonist, prazosin [113,117], but notby the a2 blocker, idazoxan. On the other hand, cloni-dine, an a2 adrenoceptor agonist suppressed the reflexin anesthetized cats [118,587]. The noradrenaline upta-ke blocker, tomoxetine, produced a slight inhibitionalone and only a slightly greater inhibition after prazo-sin [586]. However, it greatly facilitated the reflexwhen given after idazoxan, implying that the a2 adre-noceptor-dependent inhibitory mechanism is the domi-nant adrenergic modulator of this reflex under theseconditions. Thus, a1 adreneoceptor stimulation facili-tates sphincter reflexes, while a2 adreneoceptor stimu-lation inhibits sphincter reflexes. Stimulation of 5HT2serotonin receptors also facilitates sphincter reflexes[115]. The pudendal nerve reflex firing in chloraloseanesthetized cats was abolished by intrathecal adminis-tration of the k-opioid agonist, ethylketocyclazocine,leaving a hindlimb reflex and bladder activity unaffec-ted [114]. By contrast, the d-opioid agonist, D-Ser2-Leu5-enkephalin-Thr6 (DSLET), abolished bladderactivity and reduced the sphincter reflex to about 60%of control, leaving a hindlimb reflex unaffected [116].Thus the spinal opioid modulation of the external ure-thral sphincter has characteristics quite different fromthose regulating the bladder. In conclusion, the relativeselectivity of monoaminergic and opioidergic modula-tion of this pathway offers some possibility for drugdevelopment.

3. LUMBAR SYMPATHETIC OUTFLOW

Sympathetic PGN which are located in the intermedia-

te grey matter of the rostral lumbar spinal cord havesimilar morphological and chemical properties to thoseof the parasympathetic nucleus. However, the reflexconnections of the sympathetic neurones are very diffe-rent from those of the parasympathetic system. Thereare at least two types of sympathetic postganglionicneurones that appear to be involved with the regulationof motility in the hypogastic nerve, which innervatesthe bladder, as well as vasoconstrictor neurones. Theseneurones affect transmission through the pelvic gangliaand may have direct effects on the smooth muscle andurothelium. They are regulated by afferent inputs fromthe bladder and the somatic domain, and by descendingpathways from the brainstem and higher centres. In thechloralose-anesthetized cat, prazosin or doxazosin, a1adrenoceptor antagonists, suppressed spontaneousfiring or the reflex discharge recorded on the hypogas-tric nerve in response to pelvic nerve afferent stimula-tion [113,587]. Clonidine, an a2 agonist also sup-presses the pelvic-to-hypogastric reflex [587] whereasidazoxan, an ?2 antagonist, had no effect. On the otherhand, tomoxitine, a noradrenaline uptake blocker, inhi-bited the reflex, implying that the inhibitory (presuma-bly a2 adrenoceptor-dependent) mechanism was notvery active under control conditions but could be exag-gerated by elevating endogenous noradrenaline levels.This inhibition could be overcome by bladder disten-sion. These results suggest that the lumbar sympatheticoutflow is controlled by a1 excitatory and a2 inhibito-ry mechanisms. Although noradrenergic modulation ofthis pathway has been demonstrated in animals, it is notclear whether manipulation of the lumbar sympatheticoutflow to the urinary tract can be exploited therapeuti-cally.

1. LOCALISATION AND HISTOCHEMISTRY

The organisation of the peripheral autonomic nervoussystem supplying the lower urinary tract is not wellunderstood and is probably considerably more compli-cated than suggested by the classical picture of para-sympathetic and sympathetic innervation shown inmost medical text-books. It is now clear that many per-ipheral ganglia supply post-ganglionic input to thevarious elements of the lower urinary tract, but it is dif-ficult to define these ganglia unambiguously as eithersympathetic or parasympathetic. Apart from the pelvicplexus, itself a diffuse and ill-defined area of nerves andganglia, autonomic ganglia are present in the bladder,both in the suburothelium and the smooth muscle

V. PERIPHERAL AUTONOMICINNERVATION AND GANGLIA

99

layers, and in the serosa surrounding the bladder neckand proximal urethra. An example is shown in Figure10. A similar pattern is seen in many mammals,although the rat has a more discreet pelvic ganglion andfew, if any ganglia are present in the bladder wall itself[119].

Recent studies have looked in some detail at the histo-chemistry and immunohistochemistry of nerves andganglia supplying the human lower urinary tract. Pan-neuronal stains such as PGP 9.5 can show the fullextent of the local innervation and percentages of thenerves or neurones containing the various transmitters,neuromodulators or enzymes involved in synthesisingthem can be assessed using specific staining tech-niques. Acetylcholinesterase is most commonly used toidentify putative cholinergic nerves and NADPH dia-phorase to localise NO synthase containing nerves.Immunoreactivity to tyrosine hydroxylase (TH), neuro-peptide Y (NPY), vasoactive intestinal polypeptide(VIP), calcitonin gene-related peptide (CGRP) substan-ce P (SubP), enkephalin (ENK), somatostatin (Som),galanin (Gal), neurokinin A (NKA), nitric oxide syn-thase (NOS) and haemoxygenase (HO-2), synthesisingCO, have been investigated and shown to be present innerves running in the lower urinary tract. Choline ace-tyltransferase (ChAT) immunoreactivity has also beenstudied, but it has proved difficult to get reliable stai-ning of nerves using antibodies to this enzyme; morerecently the distribution of vesicular acetylcholinetransporter (VAChT) has been used to identify choli-nergic nerves and neurones [120].

a) Innervation of the bladder body

There are several published studies looking at the dis-tribution and co-localisation of markers in nerves in thenormal human bladder, and Table 4 shows the results of

one study looking at co-localisation of two markers innerves in the bladder wall [121]. Sensory and motornerves are present. Evidence suggests that SubP, CGRPand NKA are markers for sensory nerves [54,122]. Inthe human bladder, these markers occur infrequently innerves running in the detrusor but are moderately fre-quent in the suburothelial layer [55,56,57,121]. There isclearly more than one population of CGRP immuno-reactive nerves. A minor population also contains SubPand NKA, and these are suggested to be extrinsic sen-sory neurones (with their cell bodies in the dorsal rootganglia), but another population of nerves are found inwhich CGRP co-localises with Gal and NPY [57,121].

The majority of nerves running in the detrusor stainpositively for acetylcholinesterase and for VAChT[120,122] and are presumptive post-ganglionic para-sympathetic nerves. They run within the musclebundles parallel with the cells and show prominentvaricosities. Most of these nerves are also positive forboth NPY and VIP; smaller populations show immuno-reactivity to NOS and to Gal. Putative postganglionicsympathetic fibres immunoreactive for TH and NPYare seen only rarely in the detrusor, although they aremoderately frequent in the suburothelium [121]. Choli-nergic nerves are also present in the suburothelium;most of them in addition contain NPY and some containNOS. Their function is uncertain, although in otherorgans they are thought to play a secretomotor role[120].

Neuronal cell bodies in the bladder wall ganglia areagain a heterogeneous population. The ganglia arefound throughout the bladder wall and can be small,consisting of only one or a few nerve cell bodies, orlarge with more than 30 neurones [57,123]. They showimmunoreactivity to VIP, NOS, NPY and Gal invarying amounts. These neurones presumably synthesi-se acetylcholine, although there is a scarcity of infor-mation about ChAT immunoreactivity in human blad-der. Recent results from Dixon et al (2000) [120] showthat in tissue from children or neonates, 75% of theintramural ganglia show immunoreactivity to VAChT,95% to NPY and 40% to NOS. VIP was not looked atin this study. Intramural neurones do not show immu-noreactivity to ENK, SubP, CGRP or Som [57] sugges-ting that cell bodies of sensory neurones are not locatedhere. Immunoreactive profiles of nerves synapsing onintramural ganglia also show distinct classes. Bothpopulations of CGRP-containing nerves synapse onthese ganglion cells - the SubP/CGRP nerves presuma-bly being collaterals from the extrinsic sensory nerves.Postganglionic sympathetic nerves containing TH andNPY also synapse on these neurones, and the othergroup is the VIP/Gal/NPY containing nerve terminals,which are presumably the presynaptic cholinergicinputs. Nerve terminals immunoreactive to NOS or

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Figure 10 : Preparation of guinea-pig autonomic gangliafrom bladder neck region, stained with NADPH diaphora-se. Note large ganglion with many inputs. A sub-populationof the cells stains positively, showing NOsynthase contai-ning neurones.

Som do not appear to synapse with intrinsic neurones inthe bladder wall.

b) Innervation of the bladder neck, trigone andurethra

Autonomic ganglia are also found in the serosa aroundthe proximal urethra and prostate, and in the walls ofthe bladder neck, trigone and in the female, the proxi-mal urethra. In the human male, however, no gangliaare found in the walls of the urethra. The bladder neck,trigone and urethra can be shown to receive a morecomplicated innervation than the detrusor, with functio-nal evidence for noradrenergic and cholinergic predo-minantly excitatory innervation of the smooth muscle,and also non-cholinergic, non-adrenergic inhibitoryinnervation [124]. Histochemical and immunohistoche-mical examination of the ganglia and nerves againshow a heterogeneous population. Table 5 gives infor-mation about paraurethral ganglia in the adult humanfemale and co-localisation of various markers [125].Putative sympathetic ganglia demonstrating TH immu-noreactivity were common, and there was co-localisa-tion with NPY (» 80%), VIP (» 70%), and NOS (»50%). Similar co-localisation has been seen in maleinfants [126], and this study also showed co-localisa-tion of TH with CGRP (» 50%) and Som (» 70%). Inthese male infants, ganglia not containing TH the majo-rity showed immunoreactivity to NPY and Som (»90%), others showed immunoreactivity to CGRP (»65%), NOS (» 45%) and VIP (» 40%). In the proximalfemale urethra, virtually all of the NOS immunoreacti-ve cells were also immunoreactive to HO-2, but of theHO-2 positive cells, 25% did not show NOS immuno-reactivity [127].

c) Conclusions

The above work strongly suggests a considerabledegree of complexity of the peripheral autonomic path-

ways controlling the lower urinary tract. Although themajority of the ganglia in the bladder wall appear to beparasympathetic, at least three classes of neuronesynapse onto these cells, giving considerable scope forcomplex control. In the para-urethral ganglia it isimpossible to classify any particular ganglion as sym-pathetic or parasympathetic, or to designate all the NOScontaining ganglia into one or other of these classes.The situation resembles in complexity the entericplexuses found in the gut wall, and it is impossible atpresent to rule out the presence of local circuits andreflexes as well as the extrinsic control coming from thecentral nervous system. More work is clearly needed inthis area.

2. ELECTROPHYSIOLOGICAL STUDIES ON

GANGLIA

Methods have been developed by Hanani and Maudlej[128] for visualising living ganglion cells in the guinea-pig bladder wall, and recording their electrical proper-ties. Recently this method has been extended to theparaurethral ganglia in the same animal. Figure 10shows one of the paraurethral ganglia stained withNADPH diaphorase, showing a ganglion containing alarge number of neurones, some of which stain positi-vely showing the presence of NOS. Preparations likethis allow electrical recordings of the responses of theseganglion cells to stimulation of different preganglionicnerve fibres. Activation of most inputs results in theproduction of excitatory junction potentials that areblocked by nicotinic-receptor antagonists. Preliminarystudies show that many of these cells appear to possessSubP receptors, activation of which enhances the sensi-tivity of the cells apparently through a reduction in themembrane conductance (Figure 11).

3. INTERSTITIAL CELLS

Over the last few years, growing attention has been

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Table 4 : Results of double immunolabelling of ganglia in sections cut from the proximal urethra of 4 human females.From Ho [125]

No of ganglia No of cell bodies % showing both % showing one % showing theexamined markers marker only other marker only

PGP & NOS 7 126 58 PGP 42

TH & NOS 8 113 43 TH34 NOS 23

HO-2 & NOS 8 152 74 HO-2 25 NOS 1

VIP & NOS 6 77 69 VIP 19 NOS 12

NPY & NOS 4 68 56 NPY 37 NOS 7

NPY & VIP 4 26 69 NPY 4 VIP 27

NPY & TH 6 100 83 NPY 1 TH 14

VIP & TH 8 117 57 VIP 9 TH 34

paid to a cell type in the walls of the urinary tract thathas previously received little attention, and that is themyofibroblast. Myofibroblasts are ubiquitous. They arefibroblastic cells with smooth muscle-like characteris-tics, and many postulated functions. They are involvedin such things as growth and differentiation of tissues,formation and repair of the extracellular matrix, woundhealing, secretion of growth factors and inflammatorymediators and generating the pacemaker currentsunderlying slow wave activity in gastrointestinal smoo-th muscles. Myofibroblasts have characteristic morpho-logical features. The cells are thin and often branchedor stellate. The cell membrane shows many caveolae,intermediate fibres and smooth muscle actin microfila-ments are often seen in the cytoplasm but myosin is not

present. The cells have well developed Golgi apparatus,and the cytoplasm is rich in endoplasmic reticulum andmitochondria. The basal lamina is poorly developed.The myofibroblasts are often linked together throughgap and adherens junctions.

Many different types of myofibroblast have been dis-tinguished by their particular locations, morphologicalfeatures and staining properties. In particular classifica-tion on the basis of the cytoskeletal proteins present hasbeen used, with immunostaining techniques. Threecommonly studied proteins are vimentin (V), desmin(D) and a smooth muscle actin (A). Possibly the bestknown myofibroblasts are in the gastrointestinal tract,where they are known as Interstitial Cells of Cajal(ICCs). There are several types of ICC in the gut: themain pacemaking cells form a network close to themyenteric plexus and another one close to the submu-cosal surface. These cells express the protoocogene c-kit and can be specifically stained using antibodies tothe receptor tyrosine kinase that is its gene product.

Attention focused on interstitial cells in the urinary tractfollowing the elegant work of Smet et al [129] determi-ning the distribution of nitric oxide synthase containingnerves and the cellular targets of nitric oxide. The latterwas established using antibodies to cGMP to determinewhich cells responded to the nitric oxide donor sodiumnitroprusside by activation of guanylyl cyclase. Theyshowed that whereas the smooth muscle cells in the ure-

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Table 5 : Colocalization of transmitters in nerve fibres in normal bladders as assessed with confocal laser scanning micro-scopy. From Drake et al. [58,121]

Proportion colocalizing second transmitter

Transmitter Nerve site VIP NPY CGRP NOS TH Gal SP(frequency)

VIP Muscle (F) - 4 0 2 NA NA 0Suburo. (F) - 4 0 2 0

NPY Muscle (M) 4 - 1 NA 3 NA NASuburo, (M) 4 - 2 1

CGRP Muscle (I) 0 2 - NA NA 3 2Suburo. (M) 0 2 - 3 2

n-NOS Muscle (M) 3 NA NA - CA NA NASuburo. (M) 3 - 0

TH Muscle (VI) NA 4 NA CA - NA NASuburo. MI) 4 0 -

Gal Muscle (M) NA NA 2 NA NA - NASuburo. (I) 2 -

SP Muscle (VI) 0 NA 5 NA NA NA -Suburo. (M) 0 5 -

CA= close association observed, but transmitters not colocalised, NA= not assessed, Suburo= suburothelial plexus0 No colocalisation present A Absent1 1-20% VI Very infrequent 2 21-40% I Infrequent3 41-60% M Moderate 4 61-80% F Frequent5 81-100% VF Very frequent

Figure 11 : Microelectrode recording from single ganglioncell. (a) on a slow time base, showing the effects of addi-tion of substance P on the response of the cell to injectionof small depolaring current. (b) recording on a faster timebase of the response to a single pulse in the absence andpresence of substance P.

thra responded to sodium nitroprusside with a uniformincrease in cGMP levels, the smooth muscle cells in thedetrusor did not. However, a class of cells in the humanand guinea-pig bladder demonstrated an intense induc-tion of cGMP immunoreactivity by sodium nitroprussi-de, and these were interstitial cells that morphologicallyappeared remarkably similar to ICCs. This was particu-larly interesting, since the post-ganglionic parasympa-thetic neurones in the bladder wall contain nitric oxidesynthase, although there is no functional evidence of aninhibitory nitric innervation to the detrusor.

Recently the distribution and immunocytochemistry ofmyofibroblasts in the human detrusor has been exami-ned [121]. ICC-like cells were present throughout thebladder wall, both in the laminar propria and within thesmooth muscle layer. Within the detrusor they werepresent predominantly in the interfasicular planes andon the periphery of the muscle bundles. The cells weremultipolar, with thin processes that ramified over consi-derable distances, and made anatomical contact withprocesses from other ICC-like cells, smooth muscle andvaricose nerve fibres. All the ICC-like cells showedintense vimentin-like immunoreactivity, but did notshow immunoreactivity to a-smooth muscle actin. Themajority of cells were also weakly positive for c-kit,and co-localisation of c-kit and vimentin-like immuno-reactivity was seen using confocal microscopy. TheICC-like cells did not show immunoreactivity to thepan neuronal marker PGP 9.5 or the leukocyte commonantigen. The similarity between these cells in the blad-der, and ICCs in the gut suggest that they may beplaying some role in determining the ÔmyogenicÕ spon-taneous mechanical activity seen in isolated bladderstrips, and may also be involved in mediating activitybetween nerves and muscles as has also been suggestedfor ICCs [130]. It would be interesting to know whetherthere are differences in the numbers and distribution ofthe ICC-like cells in bladders from patients with acon-tractile or hyperactive bladders.

The possibility that ICC-like cells play a role in pace-maker activity in the urinary tract is has also been exa-mined in the ureter and urethra. In the guinea-pig ure-ter, ICC-like cells have been described in the renal pel-vis [131]. These cells are concentrated in the pelvi-caly-ceal junction with some in the renal pelvis. They spon-taneously discharge action potentials which have an ini-tial spike followed by a long plateau. It has been pro-posed that they act as a preferential pathway conductingand amplifying pacemaker signals to the smoothmuscle cells in the ureter.

In all species studied, urethral smooth muscle developssustained myogenic tone, and often small slow wavesof contraction are superimposed on the basal tone. Inthe rabbit, microelectrodes have been used to record theunderlying electrical slow wave activity in urethral

smooth muscles [132], which was found to be similar tothe smooth muscle of the gastrointestinal tract,although these workers were not aware of the presenceof ICC-like cells in the urethra. Following from thisobservation and the observation that ICC-like cells werepresent in the human urethra [129], Sergeant et al [133]used collagenase to disperse cells from the rabbit urethra,and were able to demonstrate the presence of ICC-likecells, and examine both their morphology and electro-physiology. Such cells represent only a small proportionof the cells present but had morphology typical of ICCs,and as shown by Drake et al [121], in contrast to smoothmuscle cells, stained strongly with anti-vimentin antibo-dy but did not contain myosin. The ICC-like cells werenon contractile, but showed spontaneous electrical acti-vity. The authors proposed that as in the gut, the ICC-likecells may be responsible for initiating the electrical slowwaves recorded from the smooth muscle cells and thatthe tone seen in the smooth muscles may depend on thefrequency of these slow waves.

The powerful combination of immunohistochemical,ultrastructural and electrophysiological identification ofICC-like cells now makes it possible to explore the beha-viour of these cells in a range of human disease condi-tions. In the gastrointestinal tract, prominent motility dis-turbances are present and evidence of involvement ofICCs in such diseases is beginning to emerge. These cellsare obvious targets for future rational and effective phar-macotherapy of gut diseases. This may also be true ofdiseases of the urinary tract. The possibility that ICC-likecells might modulate contractile behaviour in three suchdifferent systems as (1) ureters with their distinct pace-maker region initiating synchronous phasic contractionsin the smooth muscle, (2) the detrusor with little basictone but asynchronous small tonic activity, and (3) ure-thral smooth muscle is intriguing. This field is clearlynow one that is open for further research.

Table 6 is taken from the First Consultation [2] andsummarises the various drugs that act on peripheralnerves and smooth muscle together with specificexamples and their mode of action. The reader is refer-red to this chapter [2] and references 533-576, and thefollowing discussion provides an update on recentadvances in knowledge in this area.

1. SEROTONIN (5-HT) RECEPTORS

5-HT receptors are present in the bladder and in thecentral pathways controlling micturition, possiblymainly in the latter; there is some species variation inthe effects of this transmitter. 5-HT is thought to actindirectly in the urinary bladder by the stimulation of

VI. PHARMACOLOGY OF PERIPHE-RAL PATHWAYS TO THE BLADDER

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Table 6 : Drugs used for urinary incontinence and overactive bladder. This table is based on Table 4 in the First Consulta-tion and has been updated. The conclusions are based on references 533-576.

Classification Examples Pharmacological Action

Anticholinergic Atropine Inhibit muscarinic receptors reducing the response to cholinergic stimulationTolterodine Used to reduce intravesical pressure, increase compliance, raise volume Propantheline threshold for micturition, eliminate unstable contractions. Used to treatTrospium overactive bladder with or without urge urinary incontinenceDarfenacin*

Mixed Oxybutynin Blocks muscarinic receptors and directly relax smooth muscle. May possess anticholinergic/smooth local anesthetic properties.muscle relaxants

Beta adrenergic agonists FK 175* §3 adrenergic agonist relaxes detrusor in vitro. May be useful for overactivebladder with or without urge incontinence

Tricyclic antidepressants/ Imipramine Multiple sites of action including peripheral anticholinergic and norepine-monoamine reuptake Chlomipramine phrine reuptake blocker with potential net action as a1 receptor agonist. inhibitors Amitryptiline Central effects to inhibit micturition through possible blockade of norepine-

Duloxetine*` phrine and serotonin reuptake. Thus potentially useful for overactive blad -der, urge incontinence and pain disorders. Relative action at OnufÕs nucleus via 5-HT1A receptors makes duloxetine potentially useful for stress incontinence.

Alpha adrenergic agonists Ephedrine Increase bladder outlet resistance via direct stimulation of alpha 1 Midodrine receptors. Potentially useful for stress urinary incontinence.PseudoephedrinePhenylpropanolamine

Alpha adrenergic Terazosin In addition to reducing bladder outlet resistance these agents may act inantagonists Doxazosin central nervous system of inhibit micturition. Used for overactive bladder

Alfuzosin especially associated with benign prostatic hyperplasia. Limited data on Tamsulosin unstable contractions and use in neuropathic bladder dysfunction.

Afferent nerve inhibitors Lidocaine Vanilloid receptor agonists or local anesthetics. Block sodium channels orDMSO act on vanilloid receptor expressing afferents to reduce sensation or afferent Capsaicin input necessary to trigger micturition. Could raise volume threshold for Resiniferatoxin* micturition. Thus, potentially useful for pain disorders, and overactive

bladder with or without urge incontinence.

Estrogen Estradiol Direct application intravaginally may be useful sensory urgency. May directly increase bulk of urethra or enhance mucosal seal via urothelial hyperplasia or increased vasculature. May also up-regulate a1 receptors.

* Undergoing clinical trials. Not approved by regulatory agencies.

presynaptic 5-HT-4 receptors on cholinergic neuronesto release acetylcholine, which binds to muscarinicreceptors so as to increase bladder contractility [134].

The main action of serotonin however is within theCNS, where the 5-HT-1A receptor appears to be invol-ved in the regulation of micturition. In rats, 8-hydroxy-N,N-dipropylaminotetralin (8-OH-DPAT), the prototy-pical 5-HT1A receptor agonist, produced a micturitioncontraction when bladder volume was just subthresholdfor producing a physiological bladder contraction whenadministered intrathecally or intracerebroventricullary[135]. Furthermore, studies with the 5-HT 1A receptorantagonist, WAY100635, indicate that, in the rat, thiscompound can inhibit contractions evoked by bladderdistension in a dose-dependent manner [136]. Thisaction occurred only when the compound was injectedintrathecally at the level of the sacral parasympatheticoutflow and pelvic afferent input; the drug also blockedthe contractile effects of electrical stimulation of thepontine micturition centre, which suggests that themechanism of action is inhibition of tonic descendinginput to the sacral parasympathetic nucleus.

Studies of a number of 5-HT receptor antagonists inrats suggested that only the 5-HT-1A receptor antago-nists had potentially useful effects on the bladder, indu-cing an increase in bladder capacity with no derange-ment of bladder contractility [137]. Two compoundswere used intravenously (p-MPPI and methiothepin)and induced a dose-dependent disappearance of isovo-lumic bladder contractions in anaesthetized rats, and atthe highest doses, the dose-response curves were bell-shaped. The amplitude of bladder contractions was notmarkedly altered. A consistent increase in bladder capa-city was seen with p-MPPI and methiothepin, but onlymethiothepin decreased the voiding pressure.

In cats, it appears that 5-HT1A receptor stimulation isinhibitory to micturition contractions evoked by blad-der distension [588]. In other words, the exact oppositeeffects seen in rats. 8-OH-DPAT increased the bladdervolume threshold for inducing a contraction (i.e.increased bladder capacity) but did not inhibit theamplitude or duration of the contraction once the thre-shold was exceeded. Thus, these authors concluded thatthe site of action was on the sensory limb of the reflex.The inhibitory effects of 8-OH-DPAT were most mar-ked when the bladder was irritated by infusion of a dilu-te (0.5%) solution of acetic acid. In contrast to the inhi-bitory effects of 8-OH-DPAT on bladder activity, it faci-litated striated sphincter activity under conditions ofacetic acid infusion into the bladder. Thus, 5-HT1Areceptor activation has opposite effects on bladder andsphincter activity that is evoked by bladder nociception,i.e. inhibition of bladder activity and facilitation ofsphincter activity. The 5-HT1A receptor antagonistWAY100635 blocked both effects.

Other 5-HT receptors are also involved in central regu-lation of lower urinary tract function. Steers et al .(1992) [138] reported that 5-HT2C receptor agonistsinhibit bladder activity. Furthermore, it was shown[139] that 5-HT3 receptor activation inhibited supraspi-nal transmission of bladder afferent activity. Theseauthors and others [140] also showed that administra-tion of the serotonin neurotoxin, 5,7 dihydroxytrypta-mine (5,7 DHT) facilitated bladder activity in cats andrats, respectively. Finally, 5-HT2 receptors have beenshown to facilitate reflexes to the striated sphinctermuscles in cat [141].

2. a1 ADRENOCEPTORS

Much attention has focused on the role of the sympa-thetic nervous system and in particular a1-adrenergicreceptors (a1ARs) which are members of the largefamily of G protein-coupled adrenergic receptors thatmediate action of the endogenous catecholamines nore-pinephrine and epinephrine. cDNAs encoding three dis-tinct a1AR subtypes (a1a, a1b, a1d) have been clo-ned, and the gene products have been expressed in cellsand characterized pharmacologically [142].

a1AR antagonists have been demonstrated to relaxprostate smooth muscle and in clinical studies arecapable of relieving outlet obstruction [143]. Molecularand contractility studies performed using human pros-tate demonstrate the a1aAR is the subtype responsiblefor mediating contraction of prostate smooth muscle[144]. It is not surprising therefore that several highlyselective a1aAR antagonists have recently beendemonstrated to increase urine flow in clinical settings.However, what is surprising is that while highly selec-tive a1aAR compounds are effective in relieving outletobstruction, they do not appear to relieve irritativesymptoms [145], which suggests that these symptomsmay be mediated by another type of a1AR. a1dARmRNA and protein has recently been identified inhuman detrusor and sacral spinal cord [146,147] someauthors have hypothesized this receptor may play animportant role in the development of irritative symp-toms associated with bladder outlet obstruction[142,148]. Hampel et al [149] described the modulationof these receptors by bladder outlet obstruction, andFigure 12 outlines the changes in the different areas ofthe bladder.

3. PURINERGIC MECHANISMS

Purinergic receptors appear not to be involved to anyextent in the normal function of the human bladder,although they are important in many animals. In patho-logical conditions of the bladder, then the purinergicmechanisms may be expressed; this topic is discussedin greater depth in the smooth muscle section. At least

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7 subtypes of P2X receptors appear in the rat bladder[62]. Clusters of purinergic receptor subunits can befound on the smooth muscle cell membrane beneathjunctional varicosities in the detrusor muscle of the raturinary bladder, and the P2X1 receptor appears to bethe major subtype in both the rodent and human [150].In the non-pregnant rat, clusters at junctional varicosi-ties are principally composed of the P2X1, 2, 3 and 5subtypes. But by day 14 of pregnancy, about 80% oftheses clusters are replaced by P2X4 and 6 junctionalclusters which also increase in density by more than80% [151].

The P2X1 was found to be the predominant purinocep-tor subtype in the human male bladder, consistent withpharmacological evidence [150]. These studies wereperformed on bladder biopsies that were obtained fromnine patients undergoing prostate surgery and controlsobtained from eight age-matched men undergoing rou-tine bladder endoscopy studies and who were asympto-matic. The amount of P2X1 receptor per smooth musclecell is greater in the obstructed than in control bladder,suggesting an increase in purinergic function in the uns-table bladder arising from bladder outlet obstruction. Inthe foetal human bladder, P2X1 receptors, the predomi-nant purinoceptor in adults, is expressed to a rathersmaller extent. During human development, however,there are also several marked developmental changes inpurinoceptor expression in the bladder, and the P2X4subtype is expressed in developing bladders at relative-ly high levels. Changes in the regional distribution of

purinoreceptors also occur during development, whichsuggests a changing role of purinergic transmission inthe control of bladder motility during fetal maturation[150,152].

Changes in purinergic receptor levels have been foundin the detrusor in human infant bladders, which haveonly low levels at 9 months or less of age but increasedto approach adult levels at 2 years [69]. The associationof P2X receptor subtypes and the parasympatheticnerves in the detrusor of humans was also studied [69];levels of P2X4, P2X6 and P2X7 subtypes in adulthumans were all at least 10-fold lower than other sub-types (P2X1, P2X2, P2X3 and P2X5). In IdiopathicDetrusor Instability, these authors found a selectiveabsence of P2X3 and P2X5 beneath all the parasympa-thetic varicosities, which was associated with a distur-bance of control of detrusor contractility in this condi-tion. It is not known whether P2X3 receptors on affe-rent terminals are affected (see earlier discussion).

4. TACHYKININS

Bladder distension is reported to exert some contractileresponses on the detrusor mediated by axonal reflexes:these sensory-motor functions of the afferent collateralsappear to involve the release of tachykinins, such asSubstance P and Neurokinin A, from the afferentendings in muscle. These effects only occur when theafferents are excited by distension and can be blockedby specific NK-2 antagonists [135].

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Figure 12 : The distribution of alpha-1 adrenoceptor mRNA and the effects of bladder outlet obstruction (modified fromreference 149).

The Cellular Biology chapter in First InternationalConsultation on Incontinence [1] was concerned with theproperties of the normal bladder and urethra. The pro-perties of four components were discussed: the smoothmuscle, the stroma, the blood vessels and the urothelium,and for the urethra we discussed the smooth and striatedmuscles and the lamina propria. In this current volume,further properties of the bladder smooth muscle, inclu-ding its metabolism, contraction and relaxation, excita-tion contraction coupling, Ca2+ handling and electricalconnectivity are discussed. In addition there is a new sec-tion on the ganglia present in the human bladder wall andparaurethral tissue, and on the interstitial cells that havebeen found submucosally and around the smooth musclebundles in the urethra and bladder.

First, to put the rest into context, we will briefly describesome of the important functional properties that the blad-der and urethra must have to fulfil their normal role.

The bladder is designed to store urine and expel it whenit is socially convenient, and the urethra to prevent lea-kage, and yet allow voiding during micturition. To storesignificant amounts of urine, the bladder wall must beextremely compliant and able to stretch and rearrangeitself to allow an increase in bladder volume withoutsignificant pressure rise. The bladder wall cannot,however be purely passive during filling, since initia-tion of a pressure rise can only occur if the shape of thebladder conforms to the minimum surface area/volumeratio possible anatomically. This constraint means thatthere must be continuous contractile activity in thesmooth muscle cells to adjust their length during filling,but without synchronous activity that would elevateintravesical pressure and prevent filling. The smoothmuscle and intrinsic nerves have to be protected fromexposure to urine by the urothelium, which itself mustalso expand readily during filling, whilst preserving itsfunction. Sensory nerves conveying information aboutthe state of the bladder can, however clearly respond tothe degree of distension of the bladder, and also bemodulated by the composition of the urine. Bladderemptying requires relaxation of the urethral smoothmuscle followed by synchronous activation of all thedetrusor, since if only part of the bladder wall contrac-ted, the uncontracted compliant areas would stretch andprevent the increase in pressure necessary for urine tobe expelled through the urethra. Inhibitory nerves arenecessary to switch off spontaneous tone in the urethra,and motor nerves have to innervate the detrusor smoo-th muscle sufficiently densely to achieve elevation ofintravesical pressure in micturition and maintain itduring emptying of the bladder.

1. SMOOTH MUSCLE METABOLISM AND ENER-GETICS

The emphasis of this section is on the relationship bet-ween lower urinary tract function and cellular metabo-lism and energetics. Effective bladder and urethralfunction is intimately related to intracellular metabo-lism [153-167] Although there is an extensive informa-tion base relating cellular metabolism to vascular smoo-th muscle function [168- 174], there is relatively littleknown regarding cellular metabolism and lower urina-ry tract smooth muscle function.

Both bladder and urethral smooth muscle function, andthe protective properties of the urothelial lining of thebladder and urethra, are dependent upon the normaldelivery of oxygen and nutrients. In fact, the integrityand ability of the urothelium to protect the detrusorsmooth muscle is extremely sensitive to ischaemia,acute over distension, diabetes, partial outlet obstruc-tion, and hypoxia [175-182]. A reduction in blood flowto the urothelium has been linked to a variety of disor-ders including interstitial cystitis, recurrent urinary tractinfections and incontinence [175,183].

Functional compartmentation refers to the observationthat specific cellular functions appear to utilize energyderived from selective sources such as glycolysis, oxi-dative phosphorylation, or cytosolic ATP hydrolysis(i.e. specific compartments) [184-6]. Bladder metabo-lism, like that of most other smooth muscle, is charac-terized by substantial lactate production under aerobic,resting conditions [154-156; 161,163-165,167]. Currentobservations are consistent with the concept that speci-fic enzymes and cellular functions utilize metabolicenergy derived from sources other than cytosolic ATP[187-190]. Briefly, the contractile response to bethane-chol (bladder body) and phenylephrine (urethra) isbiphasic in nature; it consists of an initial, rapid phasicrise in tension followed by a prolonged period of sus-tained tension. As mentioned above, the bladderÕs func-tional ability to empty is related to the sustained phaseof bladder bodyÕs response to muscarinic stimulation;in women, continence is related to the sustained ure-thral response to a-adrenergic stimulation [191-197].The sustained phase of the contractile responses are lostimmediately upon removal of oxygen from the bathingmedium, even though the intracellular concentrationsof ATP are high [189,190].

In figure 13, the effect of in-vitro anoxia on the phasicand tonic responses to bethanechol is compared to theeffect on ATP concentrations. The tonic response isreduced by over 80% at a time when the phasic respon-se and ATP concentrations are still above 80% ofcontrol. The phasic response to bethanechol and theintracellular ATP concentration are reduced at the same

B. SMOOTH MUSCLE

107

rate. In similar studies, Uvelius and Arner demonstratedthat the plateau response of the rat bladder to high K+

solutions was lost rapidly upon the initiation of anoxiaand restored rapidly upon re-oxygenation [198]

These observations are consistent with the concept thatsustained tension is supported by high energy phos-phates derived directly by oxidative phosphorylationrather than by cytosolic ATP. Further support comesfrom recent studies on substrate deprivation [199-201]).The urinary bladder, similar to all smooth muscles, hasa low glycogen concentration [156]. Thus, in the absen-ce of glycolytic substrates, the rate of oxidative meta-bolism decreases rapidly, whereas the cytosolic ATPconcentration decreases at a much slower rate. Incuba-tion of isolated bladder strips in the absence of glucose(or any other substrate) leads to an inhibition of thetonic phase (plateau phase) of the contractile responsethat is much faster in onset and much more pronouncedthan the inhibition of the phasic tension observed aftersubstrate deprivation. Additional studies demonstratethat the tonic response to field stimulation is signifi-

cantly more sensitive to glucose depletion than is thetonic responses to bethanechol or KCl; although thetonic responses to all forms of stimulation are moresensitive to glucose depletion than are their respectivephasic responses [199-201]

In studies on the response of the rat bladder to glucosedeprivation in the presence and absence of anoxia, theplateau phase of the response to repetitive high K+ sti-mulation was significantly more sensitive to both glu-cose deprivation and the combination of glucose depri-vation and anoxia than the peak response [198]. Thus inboth rat and rabbit, the sustained responses to a varietyof forms of stimulation are significantly more sensitiveto glucose deprivation, anoxia, and the combinationthan are the peak responses. Since bladder emptying isa function of the sustained response to stimulation,these isolated strip studies support the concept thatbladder emptying is significantly more sensitive toischaemia and anoxia than is the peak voiding pressure.

2. RELATIONSHIP BETWEEN DETRUSOR SMOOTH

MUSCLE AND MUCOSAL METABOLISM

Several studies on acute overdistension, partial outletobstruction, and diuresis-induced hypertrophy indica-ted that the mucosal layer of the bladder is significant-ly more sensitive to acute perturbation than the muscu-laris [177,178,180-182]. A comparison of the metabo-lism of the mucosal layer (consisting of the lamina pro-pria, urothelium, and any connective tissue and vascu-lar tissue within this layer) and of the muscularis layer[202] is summarised in Table 7.

The results of these metabolic studies demonstrate thatthe rate of glucose metabolism to lactic acid of themucosa is more than three-fold greater than that of thesmooth muscle (Table 7). The rate of CO2 productionof the mucosa is 60% greater than that of the unstimu-lated smooth muscle. The maximal activity of the mito-chondrial enzyme citrate synthase is significantly grea-ter in the mucosa than in the smooth muscle, while theactivity of malate dehydrogenase is similar in both tis-sues. In separate experiments, two components of therespiratory chain were compared in smooth muscle andmucosa. The specific activities of NADH-cytochromeC reductase (NCCR) and cytochrome oxidase are simi-lar in both tissue compartments.

The maximal activity of the enzyme creatine kinase isnearly three-fold greater in the bladder smooth musclethan in the mucosa [202], whereas the activity of ade-nylate kinase of the mucosa is over twice that of thesmooth muscle. Although the concentrations of ATPand ADP are similar in both muscle and mucosa, thelevel of creatine phosphate (CP) is over four-fold grea-ter in the bladder muscle while the level of AMP in the

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Figure 13 : Time-dependent effect of anoxia on tissue ATPconcentration, and on the phasic and tonic components ofthe response to bethanechol. Isolated strips of rabbit detru-sor were mounted in individual baths containing 30 ml Oxy-genated Tyrode's solution containing glucose. A second un-mounted strip was placed in each bath. The strips wereincubated in for 30 minutes at which time the aeration waschanged from oxygen to nitrogen for 3.75, 7.5, 15, 30, or 60minutes. At the end of each time period the unmounted stripwas removed and rapidly frozen for ATP analysis; themounted strip was stimulated with bethanechol (250 uM)and the phasic and tonic tension determined. Control expe-riments were performed in oxygenated medium incubatedfor the same time periods. * = significantly different fromcontrol, p < 0.05.

muscle is only 58% of that in the mucosa (Table 7).Creatine phosphate acts as a phosphate donor duringperiods of high metabolic activity (active contraction)and reduced oxygen delivery (ischaemia and hypoxia).The fact that the mucosa has a significantly lower CPconcentration, and lower CK activity indicates that themucosa would be very sensitive to reduced oxygendelivery. This hypothesis was tested by comparing therate of ATP and CP hydrolysis for bladder smoothmuscle and mucosa in the presence of anoxia [203].The rate of ATP and CP hydrolysis for the mucosa wassignificantly greater than the rate of hydrolysis for thesmooth muscle. This experiment confirms the fact thatthe mucosa has both a greater rate of oxidative metabo-lism, and a greater rate of high energy phosphate degra-dation than the smooth muscle compartment of thebladder.

Recent blood flow studies have demonstrated thatblood flow to the mucosa under resting conditions isseveral fold higher than blood flow to the muscularis[204,205]. This correlates very well with the greatermetabolic rate of the mucosa, and the greater sensitivi-ty of the mucosa to ischaemia and hypoxia.

3. RELATIONSHIP BETWEEN STRESS INCONTI-NENCE AND METABOLISM

Incontinence can be divided into two major aetiologies:

urge and stress incontinence. Urge incontinence ismediated primarily by involuntary bladder contractionswhich occur during bladder filling (independent ofwhether the contractions are of myogenic or neurogenicorigin). If pressure increases above urethral resistance,incontinence occurs. Stress incontinence occurs whenfactors such as sneezing or physical activity increasethe external pressure on the bladder and further distendthe bladder base area, which can give the feeling ofurgency, directly produce incontinence, and stimulateurethral relaxation, thus inducing a major incontinenceincident.

In men, urethral resistance is largely related to the pre-sence of the prostate, and an external sphincter thatcompletely encircles the urethra; active a adrenergictone plays a relatively minor role. In women, urethralresistance, and thus continence, is much more depen-dent upon a-adrenergic tone [116,206-208].

A reduction of blood flow to the urethra (in pigs) resul-ted in rapid and parallel reductions in both urethral PO2and urethral pressure to very low values [209]. Resto-ration of blood flow resulted in an immediate and fullrecovery of both urethral PO2 and urethral pressurevalues (Figure 14). These studies provide additionalstrong support for the concept that urethral tension andcontinence are directly related to urethral tension,which in turn is dependent upon a normal delivery of

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Table 7 : Metabolic Comparison of Bladder Smooth Muscle and Mucosa

Smooth Muscle Mucosa

Glucose metabolism

Lactic acid formation (:mol /g / h) 2.4 ± 0.2 8.0 ± 0.8**

CO2 formation (:mol / g / h) 0.15 ± 0.30 0.28 ± 0.3**

High energy phosphate content

Creatine phosphate (nmol / mg protein) 20.0 ± 3@0 4.9 ± 0.8**

Adenosine triphosphate (nmol / mg protein) 12.8 ± 1@4 15.0 ± 1.3

Adenosine diphosphate (nmol / mg protein) 6.5 ± 0.8 5.1 ± 0.6

Adenosine monophosphate (nmol / mg protein) 8.2 ± 1@1 15.5 ± 1.2**

Enzyme activity

Citrate synthasea (nmol /mg protein / min) 286 ± 25 421 ± 48**

Malate dehydrogenasea (nmol / mg protein / min) 464 ± 43 525 ± 60

NCCRa (nmol / mg protein / min) 406 ± 24 386 ± 58

Cytochrome oxidasea (nmol / mg protein / min) 163 ± 20 137 ± 40

Creatine kinase (nmol / mg protein / min) 582 ± 50 280 ± 20**

Adenylate kinase (nmol /mg protein / min) 50@0 ± 5.0 104 ± 10.0**

Free fatty acid content (nmol / mg protein / min) 20.5 ± 2.2 98.5 ± 15**

Endogenous lipase activity (nmol FFA (mg protein)-1 min-1) 1.75 ± 0.25 10.6 ± 1@8**

FFA - free fatty acid; NCCR - NADH-cytochrome c reductase. a - Activity in partially purified mitochondrial preparation; Alldata are given as means ± SEM; **significantly different from smooth muscle (P<0.05).

oxygen; and that urethral competency is lost rapidlyduring periods of ischaemia and anoxia.

Supporting this concept is an in-vitro study whichdemonstrates that the tonic phase of the contractile res-ponse of isolated strips of rabbit urethra to phenyle-phrine is lost during periods of anoxia, and restoredrapidly upon re-oxygenation [193]. Figure 15 displaysa representative tracing of the effect of anoxia on bothisolated urethral strip tension and intracellular free cal-cium [Ca2+]i (using a surface spectrofluorometer).Phenylephrine stimulated a rapid sustained increase intension. The rise in [Ca2+]i preceded the rise in tensionand decayed while tension was maintained. Switchingthe bathing solution from 95% O2 -5%CO2 to 95% N-5% CO2 resulted in immediate loss of tonic tension andincrease in [Ca2+]i far above physiological levels.Immediately upon re-oxygenation, tension was restoredand [Ca2+]i fell to pre-anoxia levels. These studiesdemonstrate that anoxia results in a loss in the tonicphase of the response to receptor-mediated contraction(both a-adrenergic and muscarinic) and a dissociationof contractile tension and [Ca2+]i, resulting in therelease of bound Ca2+ from intracellular storage sites.

Although there is excellent direct evidence that activea-adrenergic tension is an important factor in continen-ce in animal models, evidence in humans comes prima-rily from drug studies showing that a-adrenergic stimu-lation is an effective therapy for specific forms ofincontinence. Diokno and Taub reported that ephedrinesulfate was an effective treatment for sphincteric incon-tinence in 27 out of 38 patients [210]; norephedrineadministration resulted in a significant increase in bothmaximal urethral pressure (MUP) and maximal urethralclosure pressure (MUCP) [211]. Several objective cli-nical studies have demonstrated that the administrationof phenylpropanolamine (similar to ephedrine with lesscentral stimulation) was an effective therapy for stressincontinence and sphincteric incontinence [212,213].Thus, there is a solid base of clinical information whichdemonstrates that a-adrenergic stimulation (especiallyvia chronic slow-release therapy) increases both MUPand MUCP and significantly reduces both stress incon-tinence and sphincteric incontinence.

1. TENSION GENERATION

The basic mechanism of contractile activation in detru-sor smooth muscle was outlined in a previous volume[1]. Briefly, motor nerves release acetylcholine andATP that act as excitatory neurotrasmitters.

¥ Acetylcholine binds to a muscarinic (M3) receptorand via a G-protein coupled mechanism leads to anincrease of intracellular inositol trisphosphate (IP3).This soluble second messenger subsequentlyreleases Ca2+ from intracellular stores to elicitcontraction.

¥ ATP binds to a purinergic (P2X1) receptor that actsas a ligand-gated ion channel causing cell depolari-sation, which, if great enough, opens L-type Ca2+

channels. The consequent Ca2+ influx can cause fur-ther Ca2+ release from intracellular stores by Ca2+-induced Ca2+ release.

I. EXCITATION-CONTRACTIONCOUPLING

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Figure 14 : In vivo recording of the urethral pressure andthe left femoral artery pressure on occlusion of the terminalaorta in the pig. Note that the urethral pressure falls as thearterial inflow is cut off.

Figure 15 : Representative tracing of the effects of anoxiaand re-oxygenation on the response of an isolated strip ofurethral smooth muscle to phenylephrine. Isolated strips ofurethral smooth muscle (rabbit) were mounted in a special-ly designed 1 ml flow-though chamber. A calcium probe wasplaced on the surface of the strip and connected to surfacespectrofluorometer. One end of the strip was tied to a ten-sion transducer. Contractile tension and intracellular freecalcium were monitored simultaneously. Anoxia was indu-ced by changing the bathing media from 95% O2 - 5% CO2to a bathing media equilibrated with 95% N - 5% CO2 .

In human detrusor from stable bladders acetylcholine isthe predominant neurotransmitter as contractions areabolished completely by atropine [214], whilst in mostanimal species (except old world monkeys) there is adual role for acetylcholine and ATP. What has becomeevident recently is that in a number of pathologiesaffecting the human detrusor a purinergic componentreappears providing an additional source of intracellu-lar Ca2+ to activate the contractile proteins [215,216].Several questions remain: i) why is ATP a functionaltransmitter in some bladder preparations and notothers? ii) does the reappearance of purinergic neuro-transmission in detrusor from pathological bladderscontribute to bladder overactivity?

There are several, not mutually exclusive, reasons forthe reappearance of purinergic neurotransmission inpathological human bladders:

a) the differential sensitivity of the detrusor to ATPand/or acetylcholine alters. This is unlikely, as expe-riments with isolated cells from stable and unstablehuman bladders show that the potency and magnitu-de of effect of these agonists, as judged by the rise ofintracellular [Ca2+], is similar in the two groups[217]. In other words the supersensitivity to agonistssuch as acetylcholine that is evident at a tissue level[218] cannot be demonstrated at a cellular level.

b) Acetylcholine and ATP are hydrolysed in the synap-tic cleft by acetylcholine esterase (AChE) and anectoATPase respectively [219]. In principle a decrea-se in the activity of either enzyme would prolong thelifetime of the transmitter and thus enhance themagnitude of the response and increase the potencyof the agonist. For example, application of the AChEinhibitor physostigmine enhances nerve-mediatedcontractions [214]. One study has indicated thatAChE activity may be decreased in neurogenic blad-ders, which are often associated with unstable activi-ty [220].

The ectoATPase present in detrusor may be inhibitedby the agent ARL 67156 (formerly FPL 67156)[221]. Application of ARL 67156 to guinea-pig blad-der strips has also been shown to increase the magni-tude of nerve-mediated contractions [222], indica-ting that the action of ATP on the muscle cell is atte-nuated by its extracellular hydrolysis. Moreovernon-hydrolysable ATP analogues, such as a,bmethylene ATP have a much greater potency thanATP itself in generating contractions. Atropine-resis-tant contractions in detrusor from unstable and obs-tructed bladders therefore may be caused by a redu-ced ectoATPase activity so that a proportion of relea-sed ATP would reach the detrusor cell membrane

c) The amounts of ATP and acetylcholine that are relea-

sed from motor nerve terminals are different indetrusor from stable and unstable bladders. Alterna-tively the breakdown products of neurotransmittersmay exert variable effects on transmitter release.Pre-synaptic muscarinic (M1) receptors exert anautofacilitatory effect on acetylcholine release,mediated by protein kinase C [223,224] with a pos-sible concomitant down-regulation by M2 and M4receptors [225,226]. There is evidence that the faci-litatory mechanism at least is upregulated in animalswith spinal cord transection [227,228] and representsa further site which may enhance the overall contrac-tile state of detrusor. Less is known about the controlof ATP release in detrusor nerve terminals. Onestudy in mouse bladder has suggested that differentpresynaptic Ca2+ channels may regulate release ofacetylcholine (predominantly N-type channels) andATP (predominantly P- and Q-type channels) [229].

2. RELAXATION AND THE RELAXED STATE

Whilst considerable attention has been spent on mecha-nisms causing contraction there has been less work onthe cellular physiology of tension relaxation and of themechanisms whereby intracellular stores are refilledbetween successive contractions. These are equallyimportant topics as they will influence, for example, theoverall stiffness of the bladder wall and the ability ofthe bladder to generate consistent contractile responses.

In spite of the fact that tension generation occurs inde-pendently of membrane potential changes (and byimplication the direct involvement of ion channels) thedetrusor cell membrane has a very high density of Ca2+

channels [230]. These Ca2+ channels were believed ori-ginally to be all L-type channels, but recently it has beenshown that about 20-25% are T-type Ca2+ channels[231]. The significance of this observation is that T-typeCa2+ channels are activated at more negative membra-ne potentials, and at the detrusor resting potential (aboutÐ55 to-50 mV) a significant fraction would be open.

Continuous exposure to Ca2+ channel antagonists doesexert a negative inotropic effect on detrusor contractions[232] and when given in the interval between two expo-sures to cholinergic agonists reduces the response of thesecond exposure [233]. It has therefore been proposedthat when intracellular Ca2+ release is terminated (andrelaxation of the cell initiated), a fraction is re-accumu-lated into intracellular Ca-stores but a fraction is lostfrom the cell. The fraction that is lost is offset by Ca2+

influx through Ca2+ channels so that the cell remains ina steady-state with respect to its intracellular Ca content.This is shown in schematic form in Figure 16.

There remain a number of unanswered questions:

How is Ca2+ lost from the cell at the termination of the

111

intracellular Ca2+ transient? Possible routes include anATP-driven Ca-pump or a Na+-Ca2+ counter-exchan-ger. Recently evidence of Ca2+ extrusion through aNa+-Ca2+ counter-exchanger has been provided [234]although it is unknown if this is the sole efflux mecha-nism.

Is Ca2+ influx through Ca2+ channels regulated duringCa2+ extrusion? If so this would enhance the efficien-cy whereby the cell would return to a steady-state Cacontent.

A role for K+ channels in the regulation of detrusorfunction is suggested with respect to the regulation ofCa2+ entry via Ca2+ channels. The majority of K+ cur-rent is provided through large conductance K+ channelsregulated by the concentration of intracellular Ca2+,especially the [Ca2+] immediately below the cell mem-brane [235, 236]. A fall of the [Ca2+] would close thesechannels and so depolarise the membrane. ThesesoÐcalled BK channels are blocked by agents such asiberiotoxin and charybdotoxin. Figure 16 also shows asan inset a typical intracellular Ca2+ transient elicited by

a cholinergic agonist. The transient rise of the [Ca2+] isfollowed by a decline, and an transient undershootbelow the baseline. The transient undershoot shouldtherefore increase the closure of BK channels andhence depolarise the membrane during this phase,resulting in an increased likelihood of Ca2+ channelopening and thus enhancing Ca2+ influx. Some aspectsof this scheme have been elucidated [236], however itdoes propose a functional role for BK channel modula-tors in manipulating detrusor contractility by affectingthe efficiency of refilling intracellular Ca2+ stores.

The generation of tension within a muscle cell involvesthe interaction of actin and myosin filaments to form acrossbridge. In detrusor smooth muscle, as in mostother smooth muscles, these protein filaments are dis-persed irregularly throughout the cell. This disorgani-

II. THE CONTRACTILE MACHINERY

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Figure 16 : A schematic diagram of the cellular events concerned with contraction (left) and relaxation (right) in human detru-sor smooth muscle. To initiate contraction motor nerves release acetylcholine that binds to membrane M3 receptors. Intra-cellular IP3 (inositol trisphosphate) release Ca2+ from intracellular stores. The sarcoplasmic Ca2+ binds to calmodulin toinitiate contraction but is also removed from the sarcoplasm by re-accumulation into the Ca-stores or removed from the cellÐ possibly via Na-Ca exchange. The Ca2+ lost from the cell are replaced by a Ca2+ influx, possible through Ca2+ channels.Bottom right an intracellular Ca-transient recorded during application of a muscarinic agonists to an isolated myocyte. Notethe ÔundershootÕ of [Ca2+] after the transient .

sed arrangement results in a less rapid contraction andone not oriented necessarily along the longitudinal axisof the myocyte. However, this suits the function ofthese muscles which require a more prolonged activestate, and tension to be less directional within themuscle mass than are the cases in striated muscle.

Crossbridge cycling is powered by ATP hydrolysis butregulated by a transient rise of the sarcoplasmic [Ca2+].Ca2+ in the sarcoplasm bind to a soluble protein cal-modulin (Cm), to form a complex, Ca4Cm, which acti-vates the contractile machinery. In smooth muscleCa4Cm activates the myosin molecule via activation ofa myosin light chain kinase (MLCK), culminating inphosphorylation of the so-called regulatory myosinlight chain (RLC) [237]. RLC phosphorylation greatlyincreases actin-activated myosin MgATPase activity,which results in the heavy chain of the myosin molecu-le forming a crossbridge with an actin filament whichgenerates force. Crossbridge cycling - the continuousprocess of making and breaking of crossbridges withthe consumption of ATP - can proceed as long as myo-sin is activated by Ca4Cm, thus generating a contrac-tion. Relaxation occurs when the RLC is dephosphory-lated by a myosin phosphatase [238]. This generallyoccurs when the Ca4Cm concentration falls due to a

reduction of the sarcoplasmic [Ca2+], but other Ca2+

independent mechanisms may also stimulate RLCdephosphorylation and also cause relaxation - anexample is considered below.

Experiments with isolated contractile proteins [239]show that the Ca2+-dependent regulation of thecontractile apparatus in detrusor smooth muscle occursover a range of ionised [Ca2+] between 0.1 µM and 10µM (figure 17). This is precisely the range of [Ca2+]that is measured in the sarcoplasm of intact detrusormyocytes when they are relaxed and then activated bycontractile agonists such as acetylcholine or its ana-logue, carbachol [240] (see also figure 16). Quantitati-ve measurements such as these are important as theyreinforce the importance of modulation of the sarco-plasmic [Ca2+] as the key determinant of contractileregulation.

Although modulation of the sarcoplasmic [Ca2+] is themost important regulator of contraction in smoothmuscle such as detrusor, other factors may modulatethis process. Their significance is not fully understoodbut they offer additional routes to fine-tune the contrac-tile state of the muscle, both physiologically and phar-macologically. Such control points include phosphory-lation of MLCK itself [241] and activation of myosin

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Figure 17 : Left: the change to tension in a skinned detrusor fibre when the [Ca2+] was altered between 0.072 µM and 355µM. The fibre was ÔskinnedÕ by exposure to a solution containing a-toxin, which rendered the surface membrane freely per-meable. Blockade of the intracellular organelles left a preparation of myofibrils Ð see [27] for details. Right: plot of the ten-sion-[Ca2+] relationship from experiments show at left. The shaded area represents the range of intracellular [Ca2+] gene-rated in an intact cell during exposure to a cholinergic agonist.

phosphatase [242], both of which depress myosinATPase activity and hence contraction. Phosphoryla-tion of MLCK suppresses its own activation by Ca4Cmand activation of myosin phosphatase would reduceRLC phosphorylation.

An example of such modulation is given by the cyclicnucleotides, cAMP and cGMP, both of which relaxsmooth muscle [243]. Cellular cAMP levels are increa-sed either by increasing the activity of adenylyl cyclase(e.g. by b-receptor activation) or by phosphodiesteraseinhibition (e.g. application of caffeine). Addition of caf-feine to a detrusor cell gives a good example of the rele-vance of the cyclic nucleotide-dependent mechanisms.Caffeine evokes a substantial rise of the [Ca2+]i as itcan evoke near maximal release of Ca2+ from intracel-lular stores; but paradoxically it generates little contrac-tile activity [244], presumably by increasing cellularcAMP. cAMP activates a kinase (protein kinase A,PKA), and it was believed that PKA would mediaterelaxation by for example phosphorylating MLCK[245], or myosin phosphatase. More recently it hasbeen proposed that cAMP can also activate proteinkinase G (PKG - the kinase activated by cGMP), and itis by this route that relaxation is mediated. The precisedetails remain to elucidated in lower urinary tractsmooth muscles.

¥ INTERCELLULAR COUPLING

The importance of intercellular coupling in detrusorsmooth muscle function remains an unresolved ques-tion. Connection between the sarcoplasmic space ofadjacent cells is achieved by gap junctions. These aregenerated by the fusion of two pores in the surfacemembranes of contiguous cells to form a continuoussarcoplasmic bridge. The pore consists of members of aprotein family called connexins and the size of the pore,and hence its physical properties, is determined by theparticular connexins present [246].

In detrusor, many investigators using transmission elec-tron microscopy have been unable to observe gap junc-tions, or see them only very occasionally [247], whichhas led to the conclusion that detrusor smooth musclecells are not electrically coupled to a significant degree.It has been proposed that unstable activity in the blad-der may be facilitated by enhanced coupling in this tis-sue [218]. In a subset of patients with detrusor overac-tivity a microscopical study showed that samples fromtheir bladders had a greater number of so-called protru-sion junctions, indicative of intercellular coupling[248]. However, more recently connexin proteins havebeen visualised in normal detrusor (dome and trigone)using immunohistochemical techniques or scanningelectron microscopy [249,250]. However, their concen-tration is low and they are not clustered together, which

may account for the difficulty in observing them withmore conventional techniques. The immunohistoche-mical studies also show that the connexin subtype isconnexin45 which forms relatively low conductancepores, rather than connexin 43 which is found, forexample, in the myocardial intercalated disk.

These microscopical studies are in reasonable agree-ment with electrophysiological data. Experiments inwhich relatively large currents are passed throughdetrusor preparations demonstrate some passage of cur-rents between several cells indicating some degree ofelectrical coupling [251,252]. However, the magnitudeof coupling is much lower than for example in myocar-dium which corroborates the histological observations.Electrophysiological experiments using small currentsinjected into cells via microelectrodes have been unableto demonstrate significant coupling [253]. This mayresult from the relatively high resistance between adja-cent detrusor cells which would limit the passage ofsuch small currents, or the possibility that the detrusormass is not a homogeneous medium but may consist ofdiscrete and isolated bundles. The experiments passinglarge currents would lack the resolution to resolve anyheterogeneity unlike those using microelectrodes.

The implications of these observations are important tounderstand why the detrusor muscle mass may behaveas a functionally-coordinated tissue. If intercellularcoupling is significant this will allow electrical signalsto pass from cell to cell. In human detrusor, electricalevents associated with contractile activation (mediatedby purinergic transmitters) would be evident especiallyin tissue from patients with obstructed or unstable blad-ders [216,217]. Thus signals generated in one part ofthe bladder may propagate to other regions and thuscontribute to the generation of an unstable contraction.The speed at which electrical signals travel, dependsupon the actual magnitude of the coupling resistance:the higher the resistance the slower is the propagationvelocity [254]. Approximate calculations show that anaction potential could propagate at a velocity of about 5cm.s-1 [252]. This is probably too slow to coordinatethe entire human bladder during a voiding contractionbut could be sufficient to generate local contractions ora more slowly-developing contraction associated withabnormal bladder activity.

Gap junctions do not permit the passage of only electriccurrents but also of small molecules up to several hun-dred Daltons weight to ensure chemical coupling bet-ween cells. The importance of such cell-to-cell transfe-rence in harmonising the biochemical behaviour ofdetrusor is an unexplored subject.

¥ SUMMARY

Abnormal contractile activity of tissues in the lower

114

urinary tract remains an important cause of adversesymptoms. The need to study the cellular physiology ofthe component tissues remains in order to understandthe normal mechanisms that determine contractile acti-vity as well as develop more specific agents that cantarget better lesions. Whilst our understanding of theindividual cells that comprise the lower urinary tract isalways improving, a number of themes are emergingfor future work:

1. to understand in more detail the cellular pathwaysthat determine and modulate the generation of forcein lower urinary tract smooth muscle

2. to define the interaction between different cell typesthat may modulate each others function.

3. to identify more specifically proteins that are up- ordown-regulated in abnormally functioning bladdersand correlate these with changes to function

4. to investigate the precise transduction mechanismsunderlying the sensory responses evoked the blad-der, and the influence of surrounding tissues on thefunction of efferent nerves.

Normal micturition is controlled by neural circuits inthe brain and spinal cord that coordinate the activity ofvisceral smooth muscle in the urinary bladder and ure-thra with activity of striated muscle in the urethral

sphincter [4-6,8, 585]. These circuits act as on-offswitches to shift the lower urinary tract between twomodes of operation: storage and elimination. In infantsthese switching mechanisms function purely in a reflexmanner to produce involuntary voiding; however, inadults urine storage and release are subject to volunta-ry control, presumably mediated by the cerebral cortex.Thus, the neural control of the urinary tract is distinctfrom that of other visceral organs such as the heart,blood vessels and the intestine that are regulated exclu-sively by involuntary (autonomic) reflex mechanisms.

Injuries or diseases of the nervous system in adults aresome of the causes of disruption of the voluntarycontrol of micturition causing the re-emergence ofreflex micturition, resulting in bladder hyperactivityand incontinence (Fig. 18) [4-7]. Because of the com-plexity of the central nervous control of the lower uri-nary tract, incontinence can occur as a result of a varie-ty of neurological disorders as well as changes in theperipheral innervation and smooth and skeletal musclecomponents.

The reflex circuitry utilised by the micurition reflex in

the adult includes (1) parasympathetic pre-ganglionicneurones originating in the sacral parasympatheticnucleus (SPN), (2) somatic motoneurones innervatingthe external urinary sphincter (EUS) which appear tohave reciprocal activity compared with SPN efferentneurones, (3) ascending and descending neurones thatconnect sacral primary afferents and the SPN with thethe Pontine Micturition Centre (PMC), (4) and primaryafferents from the lower urinary tract. There are alsoother central pathways that are not essential for the mic-turition reflex, but influence it and modulate its excita-bility. Most of the latter act by altering the transmissionacross synapses of the micturition reflex pathwaywithin the CNS, but some also act through the lumbarsympathetic outflow.

1. SACRAL PARASYMPATHETIC NUCLEI, PUDEN-DAL NERVE (ONUFÕS) NUCLEI AND THEIR

INTERNEURONES

In the spinal cord, afferent pathways terminate onsecond order interneurons which relay information tothe brain or to other regions of the spinal cord includingthe preganglionic and motor nuclei. Because bladder,

I. SACRAL SPINAL CORD

C. CENTRAL PATHWAYS THATCONTROL MICTURITION

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Figure 18 : Influence of postnatal maturation and patho-logy on voiding function. In infants, voiding is initiated andcoordinated by reflex circuits. In older children and adultsafter maturation of central neural pathways voiding iscontrolled voluntarily by neural circuitry located in highercenters in the brain. A defect in neural maturation (redarrow) can allow involuntary voiding to persist in adults.Diseases, neural injury or aging can disrupt the centralneural pathways mediating voluntary control of micturitionand lead to the reemergence of primitive reflex mechanismsthat were present in the infant or that appear as the resultof synaptic remodeling and the formation of new reflex cir-cuitry. The goal of therapy is to reverse pathology-inducedinvoluntary micturition and to reestablish normal volunta-ry control of voiding.

urethral and sphincter reflexes are mediated by disy-naptic or polysynaptic pathways but not by monosy-naptic pathways, interneuronal mechanisms must playan essential role in the regulation of lower urinary tract(LUT) function.

Electrophysiological [36,255-7] and neuroanatomicaltechniques [258-261] including trans-synaptic tracingof pathways with pseudorabies virus (PSV) and theexpression of the immediate early gene, c-fos (Fig.5a,b) have identified LUT interneurons in the sameregions of the cord that receive afferent input from thebladder (Fig. 5a). There is agreement between thesetechniques that interneurones concerned with bladderand/or urethral sensation and reflexes are located in theregion of the sacral parasympathetic nucleus (SPN), thedorsal commissure (DCM) and the superficial laminaeof the dorsal horn (Fig. 5c).

The micturition reflex can be modulated at the level ofthe spinal cord by interneuronal mechanisms activatedby afferent input from cutaneous and muscle targets aswell as inputs from other visceral organs [4,6,256, 262-4]. A potential site for modulation is at the first synap-se in the reflex pathway between the bladder primaryafferent and the second order projection neurons inlamina I and/or the intermediate gray matter[256,262,265,267] , a region known as the Lateral Col-lateral Pathway (LCP), which is likely to be one of themost important sites for the coordination of storage andvoiding reflexes. Stimulation of afferent fibers fromvarious regions (anus, colon/rectum, vagina, uterinecervix, penis, perineum, pudendal nerve) can inhibit thefiring of sacral interneurons evoked by bladder disten-sion [256]. This inhibition may occur as a result of pre-synaptic inhibition at primary afferent terminals or dueto direct postsynaptic inhibition of the second orderneurons. Direct postsynaptic inhibition of bladder PGNcan also be elicited by stimulation of somatic afferentaxons in the pudendal nerve or by visceral afferentsfrom the distal bowel [266,267]. The neurons concer-ned contain a plethora of neurotransmitters and recep-tors that have been shown to inhibit afferent processingin somatic systems [5,6], and these will be consideredbelow. Suppression of overactive bladder and of urgeincontinence in patients by sacral root stimulation orother forms of neuromodulation may reflect in part acti-vation of the afferent limb of these viscero-bladder andsomato-bladder inhibitory reflexes[268-270]. Indeed,electro-stimulation of the pelvic nerve triggers changesin the sacral cord including cfos expression and altera-tions in neuropeptide immunoreactivity [271] .

These regions of the sacral cord also receive inputsfrom multiple descending supraspinal systems; e.g.from the pontine micturition center [36,272], paraven-tricular hypothalamus [273] , serotonergic raphe spinal

neurons [274] , and adrenergic bulbospinal neurons[107]. This convergence of numerous pathways in theLCP suggests that the processing of sensory input fromthe lower urinary tract is likely to be subjected to com-plex regulation and therefore may be susceptible tovarious neurological diseases and thus play a criticalrole in the emergence of neurogenic bladder disorders.

a) Segmental Synaptic Inputs to parasympatheticpreganglionic neurones (PGN)

In the neonatal rat spinal cord interneurons in the SPNand DCM regions make direct synaptic excitatorycontacts with parasympathetic preganglionic neurons,and some have inhibitory effects on the motoneuronesinnervating the external urethral sphincter [36, 255,257,275,276]. Interneurons dorsal and medial to theSPN make excitatory connections mediated by gluta-matergic receptors [36,255,275,277]. These interneu-rons can in turn be activated by electrical stimulation ofthe LCP region of the lateral dorsal horn which containsprimary afferent axons. However a minority of themedial neurons produce inhibitory synaptic responsesin the PGN mediated by the release of glycine andgamma amino-butyric acid (GABA) [276]. Individualneurons release both transmitters. Glycine and GABA,and GABA-A and GABA-B agonists inhibit distension-induced firing of PGN, and inhibition of glycine recep-tors increases enhanced basal activity in the pudendalnerve to the urethral striated muscle sphincter and pre-vented the reduction in this activity associated withPMC stimulation-induced voiding in cats [278, 279].Clinical studies have revealed that intrathecal admins-tration of a GABA-B receptor agonist (baclofen)increased the volume threshold for inducing the mictu-rition reflex [6].

b) Descending Synaptic Inputs to parasympathe-tic preganglionic neurones (PGN)s

As already stated, the LCP receives convergent inputsfrom a number of descending pathways that originate inthe brainstem, Including the pontine micturition center(PMC), paraventricular, raphe-spinal neurons, andadrenergic bulbospinal neurons.

Glutamate plays an important role as a transmitter atthe terminals of neurons originating from the PontineMicturition Centre that synapse on the PGN. Glutama-te exerts its affects through ionic and G-protein coupledreceptors (Fig 19). Tools that have been used to inves-tigate these actions of glutamate in the micturitionreflex include the Non-NMDA glutamatergic receptorantagonists kynurenic acid or 6-cyano-7-nitroquinoxa-line-2,3-dione (CNQX)) and 2-amino-5-phosphono-pentanoic acid (AP5), an NMDA glutamatergic recep-tor antagonist.

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In the rat, the amplitude of (a) micturition contractionselicited by bladder distension or of (b) pelvic PGN acti-vity elicited by stimulation of PMC is decreased byadministration of NMDA (N-methyl-D-aspartate) orAMPA (a-amino-hydroxy-5-methyl-4-isoxazole pro-pionate) receptor antagonists [277,280-283]. Howeverin the cat, responses evoked by stimulation of the locuscoeruleus are blocked by an NMDA antagonist but notby a non-NMDA antagonist [284]. It is not yet clearwhether this difference represents a species differenceor whether non-NMDA receptors are not significant inthe direct pathway from the pontine micturition centerto the PGN.

One of the main transmitters in the locus coeruleus isnoradrenaline, but its actions, mediated by a1 adreno-ceptors, on the direct pathway controlling micturitionremains to be clarified. The results of experimentsusing a-1 and a a-2 adrenergic antagonists vary withanaesthesia, and species, and some of these drugs arenot specific for adrenergic receptors [285-292]..

Opioid peptides have an inhibitory action on reflexpathways in the spinal cord. It is clear that activation ofspinal d- and probably m-opioid receptors but not k-opioid receptors inhibits isovolumic contractions evo-ked by suprathreshold distension of the bladder[293,294]. Inhibition of reflex bladder activity ismediated by d- receptors (cat) or m- and d-receptors

(rat) ; whereas inhibition of sphincter activity is media-ted by k-receptors [5,6]. The spinal inhibitory effect ofa d-opioid-selective agent, DPLPE, is blocked by the 5-HT antagonist, methysergide [295] ; thus the role of 5-HT in mediating the effects of spinally-administeredopioid peptides is unclear. Dynorphin-B, a k-opioidantagonist, had no effect on micturition even at 10times its analgesic dose [294].

Therefore, a number of substances may act in the spinalcord to modulate the strength of bladder contractions,and this may be a therapeutically interesting avenue topursue.

2. AFFERENT ASCENDING PATHWAYS

Ascending Interneurons, involved in transmitting infor-mation from bladder afferents to higher centers, havebeen identified [296, 297,298] by combined tracing stu-dies in which tracer substances were injected into brainstem or hypothalamus to retrogradely label spinal tractneurons and then cfos expression was used to detect neu-rons that responded to noxious stimulation of the blad-der/urethra, or by retrograde activation using electricalstimuli. The DCM, superficial dorsal horn and SPN allcontain interneurones with rostral projections that areactivated during noxious [44,299] or non-noxious stimu-lation [258] of the rat bladder and the urethra.

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Figure 19 : Glutamate serves as an excitatory neurotransmitter in ascending, descending, interneurons and possibly afferentsinvolved in micturition. Its actions are mediated by a wide array of receptors classified by mechanism of action. An increasein excitatory transmission could lead to OAB.

Pharmacology of Spinal Primary Afferents and Ascen-ding Sensory Pathways

Pharmacological experiments evaluating the effects ofdrugs on the central mechanisms controlling micturi-tion have commonly used intracerebroventricular orintrathecal administration of drugs. The interpretationof results from these types of experiments is now moredifficult, because recent data indicate that the micturi-tion reflex pathway may pass the through multiple relaystations in the brain stem and be modulated by inputsfrom various centers in the brain (Figs. 20 & 21). Thusit is impossible to determine the exact site of action ofdrugs when they are administered into the cerebral ven-tricles or sub-arachnoid space. All that can be conclu-ded from such an experimental design is that some cen-tral structure with access to micturition circuitry hasbeen affected by the drug. Within the spinal cord, theaction of such drugs may be on the sensory or motorcomponents of the reflex and within the brainstem; forexample cholinergic agonists are known to act on thesites within the pons and medulla to facilitate micturi-tion [300,301] and microinjection specifically at thelocus coeruleus mimics the effects of electrical stimula-tion at that site [5,302,303]. Details were provided byde Groat et al [2], but the main conclusions are sum-marized below.

Glutamate is an important excitatory transmitter in the

afferent limb of the micturition reflex, and mediates itseffects by means of both NMDA and nonNMDA recep-tors. This conclusion is based on studies of C-fosexpression and the transmission of afferent activity ros-trally, and the depressive effects of both NMDA andnonNMDA glutamatergic receptor antagonists[44,45,304].

The Tachykinins (Substance P, Neurokinin A and Neu-rokinin B) are structurally related peptides present insmall primary afferent neurons, and their effects aremediated by the NK-1, NK-2 and NK-3 receptor sub-types of neurokinin (NK) receptors. The neurotoxin,capsaicin, which acts on the VR-1 vanilloid receptor,depletes tachykinin stores and causes degeneration oftachykinin containing primary afferents and has beenused to dissect the role of these peptides. There hasbeen considerable interest in the role of tachykinins asmediators of the first step in the spinal circuitry concer-ned with micturition [2]. Intrathecal treatment of adultrats with intrathecal capsaicin caused a reversible blockof the micturition reflex [305]. This and other studies[306-8] suggest that substance P may play a part intransmission at the first synapse in the micturitionreflex. Intrathecal administration of the NK-1 antago-nists, RP 67580 and CP 96345, increased bladder capa-city in conscious rats without changing micturitionpressure; whereas NK-2 antagonists were ineffective[309].

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Figure 20: Diagram showing neuroanatomical distribution of brain and spinal cord nuclei involved in CNS regulation oflower urinary tract function. The primary components of the storage and micturition reflexes are shown in red and labeled ingold. The secondary (or modulatory) components are shown in pink and labeled in white.

An inhibitory modulation by opioid peptides of theafferent limb of the micturition reflex at a spinal levelhas been suggested [2]. However the effects of morphi-ne and its metabolites are dependent on species, anaes-thesia and mode of administration, and the availabledata [310,311] suggest that spinal opioid mechanismsmay be able to influence the afferent limb of the mictu-rition reflex.

In addition to putative involvement in the descendinglimb of the micturition reflex, biogenic monoaminesmay influence the ascending limb at a spinal cord level,and may indeed be tonically active. Depletion of nora-drenaline or 5-HT in the spinal cord increased micturi-tion volume in conscious rats [287]. while blockade ofnoradrenaline and 5-HT re uptake in chloralose anaes-thetised cats, did not affect micturition parametersunless the bladder was irritated [312]. On the otherhand, intrathecal administration of blockers of 5-HT1/2(methysergide) or 5-HT3 receptors (zatosetron) decrea-

sed threshold volume for perception of fullness inconscious cats [287,313] implying that descendingserotonergic pathways tonically modulate afferentinformation at sacral spinal level.

a-1 and Ð2 receptor agonists and antagonists haveeffects that are difficult to interpret [2,288,291] becau-se of concomitant effects on micturition pressure andresidual urine volume. Intrathecal administration of areceptor subtype selective antagonists reveal that a-1Aor a-1B receptors inhibit the frequency of bladdercontractions on CMGs in anesthetized rats [314].However, some a-1A receptors facilitate the descen-ding limb of the micturition reflex pathway.

Adenosine A1 (and perhaps A2) receptor activation byintrathecal administration of the agonist produced a caf-feine-sensitive increase in volume threshold inconscious rats probably because of the inhibition ofexcitatory interneurons, which are the presumed site of

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Figure 21 : Diagram of documented neuroanatomical connections of the primary and secondary components of storageand micturition reflexes. The connections of the primary components of the storage reflexes are shown in red, while theconnections of the primary components of the micturition reflexes are shown in gold. The connections of the secondary (ormodulatory) components of the reflexes are shown in white. Abbreviations:- A11 A11 group of dopaminergic neurons - A5 A5 group of noradrenergic neurons- KF Kollicke-Fuse nucleus - LC locus coeruleus - MPFC medial prefrontal cortex - MPO medial preoptic nucleus of the hypothalamus- NRA nucleus retroambiguus - NRM nucleus raphe magnus- NRO nucleus raphe obscuris - NRP nucleus raphe pallidus- ON OnufÕs nucleus - PAG periaqueductal gray- PMC pontine micturition center - PVN paraventricular nucleus- PVT periventricular thalamic nucleus - SPN sacral parasympathetic nucleus- SYM IML sympathetic inermediolateral nucleus

inhibitory action of the adenosine A1 agonist [315]. Nostudies have been reported in which the effects of ade-nosine receptor antagonists were evaluated, so the roleof these receptors in ongoing modulation of micturitionis unknown.

Intrathecally administered baclofen, a GABA analogueacting at GABA-B receptors, caused inhibition of dis-tension-evoked micturition in conscious rats whichappears to be independent of the release of Substance Por 5-HT. As baclofen also inhibits field stimulation evo-ked release of calcitonin gene-related peptide from pri-mary afferent terminals in dorsal horn slices, one pos-sible site for this action is suppression of primary affe-rent transmitter release.

Inhibitors of Nitric Oxide Synthase (NOS), given sys-temically or intrathecally, do not affect normal micturi-tion in conscious or anesthetized rats; NOS- knockoutmice also show no abnormality of micturition [316].However, the bladder hyperreflexia that accompaniesirritation with turpentine or acetic acid is amelioratedby spinal application of NOS inhibitors [317,318]. Incontrast the hyperreflexia induced by intravesical admi-nistration of capsaicin is not influenced by a NOS inhi-bitor, although the behavioral effects of the irritationare reduced [319]. In addition to irritation, obstructionof the lower urinary tract is accompanied by increasednNOS expression in the L6/S1 DRG and dorsal horn ofthe spinal cord [320]. It is believed that NO is involvedin mediating NMDA receptor-dependent effects but notthose involving NK2 receptors. The inhibitory compo-nents of the somatovesical reflex elicited by electricalstimulation of the tibial nerve are also reduced by NOSinhibition [321]. Thus neuromodulation may operatethrough NO mechanisms to reduce unstable bladdercontractions.

Various studies using transections, lesions, or chemicalor electrical stimulation indicate that the micturitionreflex is normally mediated by a spinobulbospinalreflex pathway passing through relay centers in thebrainstem (Fig. 3b) [4-6,9]. Neurons in the brain stemat or below the level of the inferior colliculus have anessential role in the control of the parasympatheticcomponent of micturition [4-6], whereas areas of thebrain above the colliculus appear to have predominant-ly inhibitory effects.

Although the circuitry in humans is uncertain, brainimaging studies have revealed increases in blood flowin the PMC during micturition (Figure 22) [322]. Inhumans it has also been confirmed that modulatorypathways exist in the caudal brainstem raphe serotoner-gic and coerulear noradrenergic pathways. Micturitionand urinary continence are controlled by involuntarymechanisms, which have to be distinguished from thevoluntary control of the pelvic floor musculature. Bothmechanisms are exerting influence on the same moto-neurons, but via completely separated pathways. PETscanning suggests that voluntary motor pathways invol-ved in control of the pelvic floor originate in the mostmedial part of the primary motor cortex [323].

Pontine micturition center (PMC). The dorsal ponti-ne tegmentum has been firmly established as an essen-tial control center for micturition in normal subjects.First described by Barrington [324] it has subsequentlybeen called ÒBarringtonÕs nucleusÓ, the Òpontine mictu-rition centerÓ [325], or the ÒM regionÓ [272,326]66,81

II. SUPRASPINAL PATHWAYS THATPARTICIPATE IN THE

MICTURITION REFLEX

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Figure 22 : PET scan of brainstem with increased activity in vicinity of pontine micturition center (PMC) during voiding.Alternatively , increased activity in L-region association with holding of urine. (after Blok et al [341])

due to its medial location. Because of its close anato-mical relationship with noradrenergic cells in the locuscoeruleus [327], some studies in cats have mistakenlyidentified the locus coeruleus as the essential region forcontrolling micturition [302]. The presence of recipro-cal connections between the PMC and locus coeruleus[327-329] (see below), and the existence of neurons inthe PMC that project to both the sacral spinal cord andthe locus coeruleus via axon collaterals (Fig. 21) [328],may explain some of the difficulties distinguishingthese two cell groups.

In addition to providing axonal inputs to locus coeru-leus and the sacral spinal cord [328-330], neurons in thePMC also send axon collaterals to the paraventricularhypothalamic nucleus (Fig. 10), which is thought to beinvolved in the limbic system modulation of visceralbehavior [330]. Some neurons in the pontine micturi-tion center also project to the PAG which regulatesmany visceral activities as well as pain pathways[276,331]. Thus, neurons in the PMC communicatewith multiple supraspinal neuronal populations thatmay coordinate micturition with other functons of theorganism. In the rat, ascending afferent input to thepontine micturition center arises primarily from projec-tion neurons in the lumbosacral spinal cord [332]; whe-reas in the cat some of the ascending input, particularlythat arising form Lamina I of the dorsal horn (the LCP)seems to be relayed through the PAG [298,299,333,334]. (Fig 21). In addition to receiving afferentinput from sacral projection neurons, the dorsal andlateral PAG neurons also receive input from the medialpreoptic nucleus of the hypothalamus (Fig. 10) [273],an area also implicated in sacral parasympatheticcontrol of the bladder (see below). Neurons in the dor-sal and lateral PAG project to the pontine micturitioncenter (PMC) of the cat [334] and the rat (Fig. 21)[335,336]. Other efferent connections of the PAG inclu-de medullary serotonergic and catecholaminergic cellsgroups which may modulate urine storage and micturi-

tion [335,336].

Many of the neurons within the PMC of the rat containcorticotropin releasing factor (CRF) and glutamate[337]. Neurons in the PMC provide direct synapticinputs to sacral PGN (Fig. 21) [338], as well as toGABAergic neurons in the sacral DCM [339]. The for-mer neurons carry the excitatory outflow to the bladder,while the latter neurons are thought to be important inmediating an inhibitory influence on EUS motoneurons[276]. As a result of these reciprocal connections thePMC can promote bladder-sphincter synergy. Studies inrats indicate that activation of bladder PGN by inputfrom the PMC can be blocked by ionotropic glutamatereceptor antagonists, suggesting that neurons in thePMC utilize glutamate as a neurotransmitter [328,329].

Interestingly, the PMC also provides a dense input tolateral lamina I in the sacral cord [272,338] that over-laps with the projections of bladder primary afferentneurons [340]. It is tempting to speculate that this over-lap of bladder primary afferent and supraspinal projec-tions provides an anatomical substrate for positive feed-forward facilitation of the micturition reflex to maintainan adequate excitatory outflow to the bladder in theface of decreasing levels of afferent activity as the blad-der empties.

1. PONTOMEDULLARY SYSTEMS

L-region. Axonal tracing in combination with physio-logical studies have identified many sites in the brainthat can modulate voiding function. In the pons a regionlocated laterally to the PMC (hence the term ÒL regionÓ) (Fig. 20) and just ventral to the brachium conjuncti-vum contains neurons that send a prominent input toEUS motoneurons in OnufÕs nucleus as well as a pro-jection to the thoracolumbar PGN (Fig 6) [15]. Bilate-ral lesions of the L region in the cat produce bladderhyperreflexia and incontinence [272].

Several positron emission tomography (PET) studieshave provided evidence that brainstem areas involvedin micturition control are comparable in cats andhumans. Healthy men and women were able to mictu-rate during scanning and showed an increased regionalblood flow during micturition in the dorsal part of thepons close to the fourth ventricle (Fig. 22) [341,342].The location of this area was similar to that of the PMCdescribed in the cat. A second group of volunteers wasnot able to micturate during scanning. They showed noactivation in the dorsal, but in the ventral pons, similarto the location of the PSC in cats. Apparently, thevolunteers in this not successful micturition group, pro-bably because they did not feel themselves in a safeenvironment, contracted their urethral sphincter andwithheld their urine, although they had a full bladderand tried to urinate. Thus, in humans, increases in bloodflow occur in the area comparable with the L-region ofcats during periods of urinary retention suggesting thatactivity of neurons in this region is involved in inhibi-tion of voiding (Fig 22) [322]

Serotoninergic raphe neurons. Bulbospinal seroto-nergic pathways also seem to be important in regulatingvoiding function. The sacral dorsal horn, SPN andOnufÕs nucleus are densely innervated by serotonin (5-hydroxytryptamine, 5HT) immunoreactive (IR) termi-

III. CENTRAL PATHWAYS THATMODULATE THE MICTURITION

REFLEX

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nals [274,343,344] and also exhibit moderate to highdensities of various 5HT receptor subtypes. Most, if notall, of the 5HT neurons that project to the spinal cord arecontained in the medullary raphe nuclei (Fig 20) [345].Following injection of the transynaptic tracer, pseudora-bies virus (PRV), into either the urinary bladder or theexternal urethral sphincter, numerous 5HT-IR neuronsare labeled in the medullary raphe nuclei; pallidus, obs-curus, and magnus; as well as in the neuropil just lateralto the pyramidal tract, the parapyramidal region[257,259-261]. Anterograde tracing studies also showdirect projections from the raphe nuclei to OnufÕsnucleus (Fig. 6) [346]. Electrophysiological studies havelocated neurons in the rostral medullary raphe (i.e.nucleus raphe magnus, NRM) that alter their firingduring bladder distension [5]. 5HT neurons in nucleusraphe magnus co-express inhibitory transmitters , GABAand enkephalins; while neurons in raphe pallidus (NRP)and obscurus (NRO) (Fig. 21) co-express thyrotrophinreleasing hormone and substance P, both of which havebeen co-localized with 5HT in individual terminals inOnufÕs nucleus [343]. Chemical or electrical stimulationof the raphe nucleus [5,347] or administration of serto-

nergic drugs [348] inhibits reflex bladder activity.

Pontine noradrenergic pathways. The noradrenergicsystem in the brain stem has also been implicated in thecontrol of lower urinary tract function [5,6,302]. Nore-pinephrine (NE)-containing terminals are prominent inthe sacral dorsal horn, SPN and OnufÕs nucleus[107,344,349]. Although the adrenoceptor subtypes onlower urinary tract neurons have not been identified itis known that a1A and a 1D receptors are regionallydistributed within the sacral spinal cord [350-353].. Inthe rat and human the majority of adrenergic receptorsare of the a 2 type. Less prevalent are the a 1 recep-tors. In the rat the a 1A predominates. In humans basedon mRNA data, the a1D is predominant. Transneuronaltracing with PRV has labelled adrenergic neurons in theA5 region and in the ventral portion of locus coeruleusand subcoeruleus as well as neurons in the Kollicke-Fuse nucleus (A7 noradrenergic cell group) followinginjection of virus into the bladder or urethral sphincter(Fig. 21) [257-261]. Electrophysiological studies sho-wed that bladder distension activates adrenergic neu-rons in locus coeruleus [331]. Neurons in the Kollicke-Fuse nucleus have been reported to project to the pon-tine mictrution center, as well as to autonomic centersin the spinal cord [331] The nucleus retroambiguus,located in the caudal ventrolateral medulla, containsmotoneurons involved in respiration and control ofabdominal musculature. In addition, this nucleus has aprominent projection to OnufÕ s nucleus [346,354]. Ithas been speculated that this projection activatessphincter motoneurons coincident with activation of theabdominal muscles during forceful expiration such ascoughing, laughing, sneezing.

2. PHARMACOLOGY OF SUPRASPINAL PATH-WAYS AND BRAINSTEM MODULATORY

SYSTEMS

Glutamate is believed to be an excitatory neurotrans-mitter in the locus coeruleus or parabrachial nucleus ofcats because microinjection of glutamate or its ana-logue DL-homocysteic acid mimics the effects of elec-trical stimulation at these sites (Figs 19 and 23)[355,356] . The presence of an inhibitory or mixedexcitatory/inhibitory response to excitatory amino acidinjection [356] may indicate that glutamate can alsoactivate local inhibitory pathways in the pons. Injectionof glutamate into the serotonergic medullary raphenuclei which have an inhibitory function in micturitionelicited only inhibitory effects [347].

Non-NMDA (N-methyl-D-aspartate) glutamatergicreceptor antagonists injected intracerebroventricularlyor bilaterally into the locus coeruleus blocked the acti-vation of locus coeruleus neurons, and the EEG arousalpattern; but an NMDA glutamatergic receptor antago-nist (AP-5) had no effect [357]. In conscious rats thebladder hyperactivity induced by middle cerebral arte-ry occlusion can be aborted by pretreatment with anNMDA antagonist (MK-801) or can be suppressed bytreatment after the occlusion [358]. More studies arenecessary to determine the specific receptors andsynapses where glutamate is functioning.

Gamma Amino-Butyric Acid GABA or GABA ago-nists apparently mediate a tonically active inhibitorymodulation of the micurition reflex at a supraspinallevel [356,358]. GABA or muscimol, a GABA-Areceptor agonist, suppressed reflex bladder activity andincreased the volume threshold for inducing micturitionin a bicuculline-sensitive manner when injected at sitesin the brainstem where electrical stimulation inducedbladder contraction. Because bicuculline (a GABAantagonist) alone stimulated bladder activity and lowe-red the volume threshold for micturition, it was conclu-ded that there is a tonic GABA-ergic inhibitory modu-lation in these region (Fig. 23) [592].

Interest in the role of dopamine in the supraspinalcontrol of bladder function, arises from the clinicalobservation that patients with ParkinsonÕs diseasewhich is caused by degeneration of dopamine-contai-ning neurons in the brain stem often exhibit bladderoveractivity [5,6,357]. Pharmacological studies howe-ver reveal that dopamine and its agonists have mixedeffects depending on the site and mode of application.In cats microinjection of dopamine into the PMC faci-liated the micturition reflex [6] (Fig 24), whereas intra-cerebroventricular administration of dopamine or a D-1dopamine receptor agonist (SKF38393) inhibited themicturition reflex [593].

The role of opioid peptides has been studied using

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focal microinjections of drugs that confirm some of theresults of icv injection of morphine and its antagonistnaloxone [293,359] have been obtained in urethane trea-ted rats [360]. Injection of D-Ala2-Met5-enkephalinami-de (DAMA) into the pontine micturition center blockedisovolumic bladder contraction, an effect antagonized bysystemic or local naloxone administration. Naloxoneinjected alone increased frequency without changingamplitude of contractions. Individual PMC neuronswhich were activated with bladder contraction were inhi-bited by fentanyl. Both µ- and d-opioid but not k-opioidreceptors can mediate this supraspinal effect [293,361],but apparently by different mechanisms [295].

Intracerebroventricular administration of purines andpurinergic antagonists in the rat has been used to deci-pher the role of ATP on supraspinal pathways involved inmicturition. Recently, investigators have found that theP2X3 receptor in the brainstem facilitates micturition[362].

Conclusion

3. HYPOTHALAMIC SYSTEMS

The paraventricular hypothalamus provides direct pro-jections to the sympathetic intermediolateral cellcolumn, SPN, OnufÕs nucleus and the LCP in laterallamina I in the sacral spinal cord [272], i.e. a regiondensely innervated by bladder primary afferent neurons[5,340]. The medial preoptic region (MPO) of thehypothalamus also appears to play a role in control ofmicturition since it projects to the PAG and to the PMC(Fig. 21) [272]. Further linkage between the medialpreoptic nucleus and micturition has emerged from cli-nical brain imaging studies in patients that have revea-led increases in regional blood flow in this region of thehypothalamus during micturition [322].

The paraventricular hypothalamus is one of the richestsources of neuropeptide neurotransmitters in the brain.Thus, it is possible that some of the peptidergic trans-mitters found in the SPN arise from the paraventricularnucleus. Oxytocin containing projections from thebrain, presumably the hypothalamus, are found in thesacral spinal cord [363]. Oxytocin may inhibit voiding(Fig 25). Intrathecal oxytocin raises the volume thre-shold for micturition on awake cystometrograms. Inobstructed rats, oxytocin can inhibit unstable contrac-tions [363]. These effects can be prevented by a selec-tive antagonist to the oxytocin receptor. Whether thistransmitter and hormone acts in the dorsal horn on affe-rent input, interneurons or directly on autonomic pre-ganglionic neurons is uncertain. Dopaminergic termi-nals which are thought to arise from dopamine-contai-ning neurons of the A11 cell group in the diencephalonhave been identified in the PMC as well as in the sacralspinal cord in the regions of visceral afferent projec-tions, the SPN and OnufÕs nucleus [364].

4. CORTICAL PATHWAYS

Although the forebrain is not essential for the micturi-tion reflex, clinical observations suggest that it isimportant for the beginning of micturition. Studies onanimals suggest that the prominent facilitatory effect of

In conclusion, glutamate may be involved as an exci-tatory transmitter in the supraspinal circuitry control-ling micturition and may be a mediator of bladderhyperactivity following neural injury, however therole of other potential excitatory transmitters needsto be examined. It is also clear that several sub-stances can exert significant modulatory influenceson these circuits (Fig. 11), and thereby have drama-tic influences on micturition. The receptors for thesesubstances therefore represent potential sites for the-rapeutic intervention (Fig. 24).

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Figure 23 : Coordination of voiding with external urethralsphincter. Descending glutamatergic input to sacral spinalcord excites bladder neurons in sacral parasympatheticneurons (SPN). Simultaneous activation of GABA neuronsinhibits sphinter neurons in OnufÕs nucleus. Conversely,during attempts at urine storage activation of GABA neu-rons providing input to bladder neurons while providingexcitatory input to sphincter neurons.

decerebration in animals with an intact neuraxis seemsto be due in large part to removal of tonic inhibition ori-ginating in the cerebral cortex [5]. Various corticalareas are labeled following injection of PRV into thelower urinary tract [257-261,357]; thus, cortical controlof voiding is likely to be complex. The prefrontal cor-tex of the rat is considered a visceromotor area, whilethe insular cortex is considered a viscerosensory area.Evidence for tonic cortical inhibition of bladder func-tion was also obtained in rats in which infarction of themiddle cerebral artery on one side produced a promi-nent decrease in bladder capacity [594]. This effect waseliminated by administering an NMDA glutamatergicreceptor antagonist, indicating that glutamatergic exci-tatory transmission is involved in the OAB [594].

In the cat, stimulation of forebrain structures, as ante-rior cingulate gyrus, hypothalamus, amygdala, bed

nucleus of the stria terminalis and septal nuclei, can eli-cit bladder contractions [366]. Although most of theseregions send fibers to the brainstem, only the hypotha-lamic preoptic area projects specifically to the PMC[298,367].

¥ STUDIES ON THE HUMAN CNS DURING MICTURITION

PET scanning studies suggests that this might also betrue in humans, because the hypothalamus, includingthe preoptic area, was activated during micturition[341]. The direct projection from the hypothalamicpreoptic area to the PMC probably conveys the ÒsafesignalÓ to the PMC to start micturition. Most of the PETscanning studies on micturition [341,342,343,368]point to two cortical areas: the cingulate gyrus and theprefrontal cortex. Cerebral blood flow in the cingulategyrus was significantly decreased during voluntary

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Figure 24 : Diagram of the central reflex pathways that regulate micturition in the cat. In an animal with an intact neuraxis,micturition is initiated by a supraspinal reflex pathway passing through the pontine micturition center (PMC) in the brains-tem. The pathway is triggered by myelinated afferents (Ad) connected to tension receptors in the bladder wall (detrusor). Spi-nal tract neurons carry information to the brain. During micturition, pathways from the PMC activate the parasympatheticoutflow to the bladder and inhibit the somatic outflow to the urethral sphincter. Transmission in the PMC is modulated by cor-tical-diencephalic mechanisms. Interruption of the latter mechanisms leads to bladder hyperactivity. In spinal cord transectedanimals, connections between the brainstem and the sacral spinal cord are interrupted and micturition is initially blocked. Inchronic spinal animals, a spinal micturition reflex emerges which is triggered by unmyelinated (C-fiber) bladder afferents. TheC-fiber reflex pathway is usually weak or undetectable in animals with an intact nervous system. Stimulation of the C-fiberbladder afferents by instillation of ice water into the bladder activates voiding reflexes in patients with spinal cord injury. Cap-saicin (20-30 mg/kg, s.c.) blocks the C-fiber reflexes in chronic spinal cats, but does not block micturition reflexes in intactcats. Intravesical capsaicin also suppresses detrusor hyperreflexia and cold-evoked reflexes in patients with neurogenic blad-der dysfunction. Glutamic acid is the principal excitatory transmitter in the ascending and descending limbs of the micturitionreflex pathway as well as in the reflex pathway controlling sphincter function. Glutamate acts on both NMDA and AMPA glu-tamatergic receptors. Other neurotransmitters that regulate transmission in the micturition reflex pathway include: gamma-aminobutyric acid (GABA), enkephalins (Enk), acetylcholine (ACh) and dopamine (DA). ACH has both excitatory and inhi-bitory effects on the pathway. (+) excitatory and (-) inhibitory synapse. Dopamine acts through D1 receptors on GABAergiccells and is inhibitory.

withholding of urine and during the urge to void. Theprefrontal cortex is active when micturition takes placeand during involuntary urine withholding. Possibly,activation of the prefrontal cortex and the anterior cin-gulate gyrus do not reflect specific involvement in mic-turition, but more in general mechanisms, such as atten-tion and response selection. The prefrontal cortex playsa role in making the decision whether or not micturitionshould take place at that particular time and place. Fore-brain lesions including the anterior cingulate gyrus areknown to cause urge incontinence [369]. The cingulategyrus influences strongly descending pathways invol-ved in the facilitation of motoneurons and interneurons.

PET scans were used to examine which brain areas areinvolved in human micturition [322]. Seventeen right-handed male volunteers were scanned during the follo-wing four conditions: 1) 15 min prior to micturitionduring urine holding; 2) during micturition; 3) 15 minafter micturition; 4) 30 min after micturition. Ten of the17 volunteers were able to micturate during scanning.Micturition was associated with increased blood flow inthe right dorsomedial pontine tegmentum, the periaque-ductal grey, the hypothalamus and the right inferiorfrontal gyrus [322]. Decreased blood flow was found inthe right anterior cingurate gyrus when urine was with-held. The other seven volunteers were not able to mic-turate during scanning, although they had a full bladderand tried vigorously to micturate. In this group, duringthese unsuccessful attempts to micturate, increasedblood flow was detected in the right ventral pontine teg-mentum, which is consistent with the idea, arising fromstudies in cats [272,326], that this area controls themotoneurons of the pelvic floor. Increased blood flowalso occurred in the right inferior frontal gyrus duringunsuccessful attempts to micturate, and decreasedblood flow occurred in the right anterior cingulategyrus during the withholding of urine. The results sug-gest that the human brainstem contains specific nucleiresponsible for the control of micturition, and that thecortical and pontine regions for micturition are predo-

minantly on the right side. The results are in accordan-ce with the results of a clinical analysis which was per-formed to determine the individual contribution of theright and left cerebral hemispheres to the frequency andurgency of micturition in 134 chronic hemiplegicpatients [342,370]. A mean frequency of micturition, 9times or more in 24 hours, was found more frequentlyin left than right hemiplegics. Left hemiplegics alsocomplained more often of urgency than did right hemi-plegics. A mean frequency of micturition of 9 times ormore in 24 hours and urgency co-existed more fre-quently in left hemiplegics than in right hemiplegics. Inthis study dealing with the chronic sequelae of stroke,frequency and urgency of micturition occurred morecommonly in patients with right hemisphere than lefthemisphere lesions.

A PET study was also conducted in adult female volun-teers to identify brain structures involved in the volunta-ry motor control of the pelvic floor during four condi-tions:

1) rest;

2) repetitive pelvic floor straining;

3) sustained pelvic floor straining; and

4) sustained abdominal straining.

The results revealed that the superomedial precentralgyrus, the most medial portion of the motor cortex, isactivated during pelvic floor contraction and the super-olateral precentral gyrus during contraction of the abdo-minal musculature. In these conditions, significant acti-vations were also found in the cerebellum, supplemen-tary motor cortex, and thalamus. The right anteriorcingu1ate gyrus was activated during sustained pelvicfloor straining.

There is general agreement on the finding that patientswith lesions in only the basal ganglia or thalamus havenormal urethral sphincter function. These patients canvoluntarily contract the striated sphincter and abort orconsiderably lessen the effect of the abnormal micturi-

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Figure 25 : Spontaneous contractile activity in detrusor strips dissected from normal (left) and idiopathically unstable (right)human bladders. Note the typical small phasic contractions of the normal detrusor strips, and the larger fused contractionsin strips from unstable bladders (From [377])

tion reflex, when an impending involuntary contractionor its onset is sensed. The patients with lesions in thecerebral cortex and/or internal capsule following a cere-brovascular accident are unable to forcefully contractthe striated sphincter under these circumstances. Thusthese patients have a profound abnormality in the cere-bral-to-corticospinal circuitry that is necessary forvoluntary control of the striated sphincter. It is intri-guing that a variety of emotional (depression, anxiety)and autonomic disorders are associated with changeswithin the prefrontal cortex. Because many of thesesemotional states are associated with OAB, it is temp-ting to speculate that neurochemical changes within thisarea of the brain have an impact on a range of voidingdysfunctions.

5. CONCLUSIONS: CNS PHYSIOLOGY AND NEU-ROPHARMACOLOGY

The storage symptoms of urgency, frequency, nocturia,with or without urge incontinence usually with fre-quency and nocturia have recently been referred to asthe overactive bladder syndrome (OAB). OAB-relatedproblems have a devastating impact on U.S. citizens in

all age groups and especially the middle-aged andelderly. In a population based European study of nearly17,000 subjects over age 40, 16.6% (for an estimatedprevalence of 22.2 million) reported symptoms consis-tent with OAB [371]. Only 36% had urge incontinence,and more women than men complained of OAB.Because these demographic groups are increasing insize relative to the rest of the population, the societalimpact of the OAB is likely to increase in the future.Despite the projected demand for treatment, manypatients are inadequately treated now. Quality of lifesurveys document that urinary frequency, urgency, andnocturia are some of the most bothersome aspects ofvoiding dysfunction. These symptoms may reflect, inpart, disturbances in how the afferent threshold for mic-turition is regulated or how sensory information isconsciously perceived. Other mechanisms for OABmay include changes in central mechanisms, efferentneural outflow, or in smooth muscle; each of these mayhave consequences for the functioning of other tissuesconcerned with micturition. In detrusor overactivity,and there is good evidence of severe changes in thefunction of smooth muscle and peripheral nerves.

Disturbances of the peripheral nerves and tissues cancause bladder overactivity, and models have been deve-loped to mimic these changes. These models are rele-vant to neurogenic and idiopathic detrusor overactivity,bladder partial denervation, lower urinary tract obstruc-tion, diabetes and interstitial cystitis. In some respectsthe division between peripheral and central causes ofoveractive bladder is artificial, but it serves as a usefuldivision so that the interactions between different tis-sues can be seen. It must be kept in mind there areimportant interactions between peripheral and centralneurons when there are lesions of one or other of these.There is now a greater appreciation that long-termevents involving growth factors leads to plasticity inneural pathways with implications for disorders of mic-turition. Neurotransmitters, prostaglandins and growthfactors, such as Nerve Growth Factor (NGF), are sub-stances that provide mechanisms for communicationbetween muscle and nerve. Disturbances in thesemechanisms can cause detrusor overactivity due to alte-rations in either bladder smooth muscle or autonomicreflex pathways. This bladder overactivity can, in turn,lead to urge incontinence. However some of the distur-bances that cause the release of nerve growth factormay also be associated with the loss of peripheral neu-rones, and regrowth or sprouting of affected neuronesmay or may not occur as a result of local NGF produc-tion.

I. PERIPHERAL MODELS OF OAB

D. MODELS OF BLADDER OVERACTIVITY

The pathways that mediate the micturition reflexhave been described: the reflex involves afferentsand efferents in the pelvic nerve, and in the normaladult, the connection between these involved ascen-ding and descending pathways that connect the sacralcord and the pontine micturition center. In some spe-cies the periaqueductal gray matter also participatesin this pathway, and in humans there is now eviden-ce of involvement of these areas as a result of PETscanning. Both glutamate and tachykinins are impor-tant in the spinal processing of afferent input fromthe bladder, possibly in different pathways.

Modulation of the micturition reflex Ð switching itoff or on in appropriate circumstances Ð is a functionof a number of important regions in the brain, ran-ging from the raphe nuclei in the medulla , throughareas of the pons and midbrain, up to the hypothala-mus, limbic system and cingulate gyrus and the fron-tal cortex.

Transmitters such as 5-HT, purines ,GABA, oxytocinand PACAP appear to modulate rather selectively thevolume threshold by actions in the sacral spinal cord.These mechanisms, too, seem to be favorable fordevelopment of novel drug therapies. Other modula-tors may also prove to be useful targets for drugs,perhaps specifically in hyperreflexia, but more workis required to clarify their significance.

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1. DETRUSOR OVERACTIVITY

Patients with an detrusor overactivity are defined uro-dynamically as demonstrating involuntary detrusorcontractions during the filling phase which may bespontaneous or provoked. These patients often share acommon set of symptoms, regardless of the aetiologyof the condition. These are urgency (when sensationsare intact), frequency, urge incontinence and nocturia.Urodynamic traces alone do not allow the determina-tion of whether the overactivity is idiopathic or is asso-ciated with neuropathy or outflow obstruction, althoughin the latter case the micturition pressure is likely to beelevated. The similarity of the symptoms suggests thatthere may be common factors underlying the overacti-vity, and in this section what is known about thechanges that result in involuntary contractions will bediscussed, and what are the possible causative factors.

a) Common Features

Examination of the physiological properties and ultrastructure of the bladder wall obtained from human oranimal overactive detrusors demonstrates commonchanges:

¥ Increased spontaneous myogenic activity

¥ Fused tetanic contractions

¥ Altered responsiveness to stimuli

¥ Characteristic changes in smooth muscle ultra structure

Examination of the peripheral innervation and the mic-turition reflex in animals and humans with overactivedetrusors also shows common changes:

¥ Patchy denervation of the bladder wall

¥ Enlarged sensory neurones

¥ Enlarged parasympathetic ganglion cells

¥ Increased effectiveness of a spinal micturition pathway.

All these common features make it very likely thatwhatever the aetiology of the condition, the underlyingcausative mechanisms are the same or very similar.These changes will be considered in more detail below.

b) Changes to the smooth muscle of the bladderwall.

¥ CHANGES IN THE UNSTABLE BLADDER

Smooth muscle strips dissected from overactive detru-sors often show increases in spontaneous contractileactivity. This has been seen in human bladder stripsfrom obstructed overactive bladders [372] and fromneuropathic unstable bladders [373]. A change in thepattern of the spontaneous activity is also seen which is

very characteristic of detrusor overactivity. Strips fromthese bladders show fused tetanic contractions (Fig.25), reminiscent of the activity typically shown by wellcoupled smooth muscles such as in the gut [374-7].Similar fused tetanic contractions are seen in bladdersfrom pigs with instability secondary to outflow obs-truction; indeed in those with well developed overacti-vity, bizarre patterns are often observed [73,78] (Fig.26).

Alterations are also seen in the responses of overactivedetrusor to stimulation with agonists and to transmuralelectrical stimulation (either direct, or via activation ofintrinsic nerves). In this case, there are differences inthe patterns seen in tissues from overactive bladders ofdifferent aetiology. In obstructed bladders there is asupersensitivity to muscarinic agonists and KCl with areduced contraction to intrinsic nerve stimulation[372,379,380]. Similar changes are seen in animalmodels (pig: [381,382]; rabbit: [383]). In neuropathicbladders from patients with spina bifida (Fig. 27),supersensitivity is again seen to cholinergic agonistsand KCl, but there is no change in the sensitivity of thecontractile response to intrinsic nerve stimulation,although the size of the response is smaller [384]. Inidiopathic detrusor overactivity, bladder strips showsupersensitivity to KCl, but not to muscarinic agonistsand there is a reduced contractile response to intrinsicnerve stimulation (Fig. 28) [377]. There is evidence thatunstable strips are more easily activated by direct elec-trical stimulation of the smooth muscle [384] (showingcontractions elicited by transmural nerve stimulationthat are resistant to the nerve blocking action of tetro-dotoxin).

At an ultra structural level, a common feature seen inthe overactive detrusor is the presence of protrusionjunctions and ultra-close abutments between the smoo-th muscle cells, features occurring only rarely in normaltissue [248]. Again development of these junctions hasbeen seen in animal models [385].

¥ SIGNIFICANCE OF THESE CHANGES

The properties of the normal detrusor suggest that thesmooth muscle bundles are not as well-coupled electri-cally as are most visceral smooth muscles. This poorlydeveloped electrical coupling accounts for the smallsize of the spontaneous contractions in detrusor strips,since it will prevent spontaneous synchronous activa-tion of all the smooth muscle cells. The lack of goodcoupling means that in vivo electrical activity will beable to control the length of individual cells without therisk of generating a rise in pressure. Dense innervationallows synchronous activation of the muscle and a risein intravesical pressure during micturition.

The changes that occur in the smooth muscle of the

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Figure 26 : Spontaneous contractile activity in a strip ofsmooth muscle dissected from the bladder of a pig with anunstable bladder due to partial bladder outlet obstruction.

Figure 27 : Concentration-contractile response curve to 10sec applications of carbachol of strips from normal humanbladders (round symbols) and bladders from patients withunstable bladders secondary to spina bifida (square sym-bols). Note the increased sensitivity of the smooth muscle tothe muscarinic agonist. (From [384]).

Figure 28 : Frequency-response curve to transmural nerve stimulation and concentration-contractile response curve to 10 secapplications of carbachol of strips from normal human bladders (circles) and bladders from patients with idiopathically uns-table bladders (triangles). Note reduction in response to nerve stimulation, but no change in sensitivity to muscarinic agonist.(From [218]).

overactive detrusor strongly suggest that the cells arebetter coupled electrically, so that spontaneous electri-cal activity will spread and initiate synchronouscontractions in many more smooth muscle cells. Thiswould account for the fused tetanic contractions seen inthe unstable bladder strips; the close abutments andprotrusion junctions seen ultra structurally may be themorphological correlates of this connectivity. In thewhole bladder, the increased excitability of the smoothmuscle combined with the greater connectivity couldresult in the generation of focuses of electrical activitywhich could spread and produce an involuntarycontraction.

Why should such changes occur in the smooth muscleof overactive bladders? Smooth muscle, along withmost innervated tissues, is capable of altering its res-ponses to changes in its pattern of activation. The typesof change that are seen in the detrusor smooth muscleare reminiscent of the changes due to experimentaldenervation [386]. It seems very likely that the changesin detrusor properties are caused by altered patterns ofactivation. This will be explored below.

c) Changes in peripheral innervation and themicturition reflex

¥ CHANGES IN THE BLADDER WALL

Another common feature of the overactive detrusor is achange in the macroscopic structure of the bladder wall.Regardless of the aetiology of the condition, sections ofthe bladder wall from unstable human bladder fre-quently show patchy denervation of the musclebundles. Some muscle bundles may be completelydenervated, whilst neighboring ones appear normal(Figs. 29) and in other areas sparser innervation is alsoseen [377,384,385,387]. A similar pattern is seen in ani-mal models (pigs: [388]; guinea-pigs: [389]; rabbit:[383]), although interestingly not in the rat [390], wherethe post-ganglionic neurones are all in the pelvic gan-

glia and not in the bladder wall. The denervated andsparsely innervated areas of the bladder wall becomeinfiltrated with connective tissue elements such as col-lagen and elastin [384,385,387], and in the completelydenervated areas in tissue from idiopathic and neuropa-thic overactive bladders, hypertrophy of the smoothmuscle cells is seen [387].

¥ CHANGES IN NEURONAL STRUCTURE

Animal experiments (carried out mainly in the rat) havedemonstrated that procedures such as spinal section orurethral obstruction, as well as leading to the develop-ment of bladder instability, also lead to an increase insize of both the afferent neurones in the dorsal root gan-glia (L6 - S1, [391]) and the efferent neurones in thepelvic plexus (after obstruction: [392,393]; after spinalsection: [394]). Similar increase in the size of the neu-rones in bladder wall ganglia in the obstructed guinea-pig has been seen.

¥ CHANGES IN THE MICTURITION REFLEX

Changes have also been seen in the micturition reflex inanimals after recovery from spinal shock following spi-nal injury. Electrophysiological measurements of thecentral delay in the micturition reflex show a shorterdelay in animals with spinal injury (for references see[395]) than in intact animals, suggesting that there hasbeen re-organisation of the micturition pathway from aspino-bulbo-spinal pathway to a purely spinal pathway.Also, activation of specific C-fibre afferents can triggermicturition in overactive bladders, but not in normalbladders. This latter observation is thought to be true inhumans, since in normal humans the ice water test (ins-tillation of the bladder with cold water), which isthought to activate specific C-fibres, does not triggermicturition, but does so in many patients with overacti-ve bladders [396,397]. In fact intravesical administra-tion of selective C-fibre neurotoxins such as capsaicinand resiniferatoxin is an effective treatment in manypatients with neuropathic overactivity [398].

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Figure 29 : Section of a normal human detrusor (A), and detrusor from a patient with idiopathic instability (B) stained to showthe presence of acetylcholinesterase on the axons of intramural nerves. Note uniform dense staining of the smooth musclebundles in normal tissue and exceptionally dense innervation of one muscle bundle adjacent to another completely denerva-ted bundle in unstable tissue (From [218]).

d) Possible causes of these changes

It seems inherently likely that the changes associatedwith the overactive bladder are the result of alterationsin the activity in the neuronal pathways controlling thedetrusor. The sensory neurones are likely to encounterincreased stimulation after outflow obstruction orinflammation of the urothelium, which could result inthe hypertrophy of these neurones and also of the gan-glia, since presumably increased parasympathetic acti-vation will result. The patchy denervation suggests thatthere has been death of some of the intrinsic neuronesin the bladder wall. The most likely cause of this isbladder wall ischaemia, which will be discussed inmore detail in the next section. In brief, the bladder hasbeen shown to be susceptible to ischaemia [124, 377,399], since anything that raises intravesical pressure tomore than 30-40cms H2O seriously compromisesblood flow in the wall. In outflow obstruction wherethere is a significant hypertrophy of the wall, increasedmetabolic demands [400] and reduced blood flow canlead to significant periods of anoxia which might resulteventually in neuronal death (Fig. 30) . This could alsoresult from detrusor sphincter dyssynergia in cases ofspinal injury.

The changes in the reflex pathway that occur after spi-nal injury probably result from the loss of descendingpathways. This may trigger the production of growthfactors and the resultant sprouting of axon terminals insurviving pathways to make new and abnormal connec-tions; for instance the C-fibres may increase theirconnections to the interneurones or motor neurones inthe segmental spinal parasympathetic pathways. Theenhanced size of the efferent post-ganglionic neuronesin obstructed bladders appears to be the result of the

liberation of nerve growth factor (NGF) by the detrusor,since autoimmunization of rats against NGF reducesthe neuronal hypertrophy [401]. Presumably the stimu-lus for the production of NGF may be the increasedwork load of the obstructed bladder [402].

c) The problem of urgency

Everyday experience would suggest that urgency is aseparate sensation from that of bladder fullness. Animalexperiments recording from afferent nerves duringbladder filling and contraction [19,20,403,404] haveshown increased activity in a uni-modal population ofsmall myelinated and unmyelinated (Ad and C) fibresin response to intravesical pressure and contraction,suggesting that there are in-series stretch receptors inthe bladder wall, whose activity correlates with the sen-sations of bladder filling and fullness. However, itseems unlikely that fibres with such properties mediatethe sensation of urgency. Again from everyday expe-rience, urgent desire to void disappears as soon as mic-turition starts - at a time when intravesical pressure andcontractions will both be high. Unfortunately the rela-tionship between urgency and detrusor activity cannotbe investigated in animals, since we cannot know whatsensations they perceive. In humans, however, it isclear from both normal and ambulatory cystometry thatinvoluntary pressure rises may occur without elicitingany apparent sensations. A sensible theory about urgen-cy has been proposed by Coolsaet et al [405]. They sug-gest that urgency is triggered by local distortions in thebladder wall, caused by activity in some musclebundles but not others. Such a condition would arise innormal bladders if a small population of low thresholdpost-ganglionic parasympathetic neurones were direct-ly activated through a spinal pathway, for instance

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Figure 30 : Urodynamic tracings from a normal pig (upper two traces) and an obstructed pig (lower two traces). Top trace ofeach shows intravesical pressure, and bottom trace oxygen tension in the bladder wall. Blood flow (not shown) follows a pat-tern that is the inverse of the pressure rise. Note that the drop in oxygen tension significantly outlasts the rise in detrusor pres-sure and drop in blood flow. (From [124]).

towards the end of bladder filling. Because of relative-ly poor coupling between bundles, such diffuse activitywould not cause a rise in intravesical pressure, butcould cause sufficient local distortions in the bladderwall to activate a population of nerve fibres that mightspecifically mediate the sensation of urgency and playa useful role in encouraging the initiation of normalmicturition. In overactive bladders, however, such dif-fuse activity might spread because of the increasedelectrical connectivity to give rise both to a prematuresensation of urgency and an involuntary pressure rise.This theory would account for the efficacy of antimus-carinic drugs in the treatment of urge incontinence. Ifonly a small number of ganglia were activated directlyby the sensory nerves, suppression of their effects couldeliminate both premature urgency and the involuntarycontractions. Normal micturition would still occur,since this would involve recruitment of many moreganglia, and direct activation of all the smooth musclecells. An increase in the sensitivity of fibres mediatingurgency may be responsible for the enhanced urgencyseen in inflammatory conditions, interstitial cystitis andother examples of sensory urgency.

f) Conclusions

It is clear that we are still some way from really unders-tanding what causes urge incontinence. However thereare sufficient clues from the observations and experi-mental results described above to suggest that we aredealing with the consequences of altered patterns ofactivation of the nerves in the micturition pathways andthus of the smooth muscle in the detrusor. In cases ofsevere urgency and urge incontinence, the process hasprobably been on-going for years, has caused periodicbladder wall ischaemia and has reached a stage inwhich neurones in the bladder wall have actually died;the smooth muscle in the detrusor has then become ableto generate synchronous contractions and these result inthe unstable pressure rises seen. What precise mecha-nism triggers these involuntary contractions is not yetclear, although it is likely that residual neuronal activi-ty of some sort is often involved.

It may be that the most likely scenario for urge inconti-nence which is associated with overactive contractionsis as follows: activity in some muscle bundles genera-ted either by stretch or by diffuse activity in parasym-pathetic fibres causes local distortions of the bladderwall, this both activates the sensation of urgency andspreads to cause synchronous activation of the bladderwall resulting in an involuntary pressure rise. Whathappens after this may vary. If the contraction is largeenough to overcome the outflow resistance, leakagewill occur. This could in its turn activate receptors inthe urethra and trigger secondary changes (transientopening of the bladder neck etc.), but often the pressu-

re and sensation of urgency will subside. Synchronousdetrusor contraction will itself reduce the sensation ofurgency and the bladder pressure may return to normal.Activation of the detrusor smooth muscle, unless sup-ported by continuous neural input, is self limiting, sincethe muscles possess Ca2+ activated potassium channelswhich ensure that the membrane hyperpolarizes andspontaneous action potentials will be switched off tran-siently [406]. Normally urge incontinence does notresult in complete bladder emptying, but if there is awell developed segmental spinal micturition reflex andloss of any descending control, activation of the pres-sure/stretch receptors may reinforce the activity andproduce bladder emptying.

g) Metabolic Studies related to unstable bladdercontractions and outlet obstruction

In man, it is difficult to investigate the cellular mecha-nisms which result in progressive bladder dysfunctionand overactivity. However, animal models of bladderoutflow obstruction can be used to mimic the functionalchanges associated with benign prostatic hyperplasia(BPH) which leads in many patients to overactivity.This pathology can be induced in rat, rabbit, and pig[407,408]. Such models can be used to examine the cel-lular and metabolic changes occurring in the smoothmuscles.

In rabbits, partial outlet obstruction induces a shift fromaerobic to anaerobic metabolism as demonstrated bythe shift in glucose metabolism from CO2 to lactic acidgeneration (Table 8). Similarly, there is a markeddecrease in the metabolism of pyruvate (mitochondrialsubstrate) to CO2 [409,410]. In the rat, lactate dehy-drogenase (LDH) activity (per mg wet weight) increa-sed with the duration of obstruction, becoming statisti-cally significant at 90 days of obstruction [409,410].

In partial outlet obstruction in rabbits, the cellularmechanism responsible for decreased oxidative meta-bolism involves decreased mitochondrial activity, asdemonstrated by specific decreases in the enzymaticactivities of citrate synthase and malate dehydrogenaseand decreased ATP synthesis [166,411-413]. However,there were no effects of outlet obstruction on the activi-ties of a variety of other enzymes including adenylatekinase, creatine kinase, hexokinase, myosin ATPÕase,and adenine phosphoribosyl transferase (Table 8) [414-416]. Further studies demonstrate that there is a dysre-gulation of mitochondrial genetic function[417].

In the rat, mitochondrial metabolism is not reduced tothe same extent as in the rabbit model [418]. Recentstudies have demonstrated that citrate synthase andmalate dehydrogenase activity of the hypertrophied ratbladder (12 days following obstruction) were reduced

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by 22% and 28% respectively when calculated per mgprotein. No significant changes were observed if theenzyme activities were calculated per mg wet weight[418]. Additional studies on the hypertrophied rat blad-der demonstrated that there was a modest reduction inthe volume-fraction of mitochondria [418,419].

h) Ischaemia as an aetiological factor in obs-tructive dysfunction

Mild partial outlet obstruction results in an immediateincrease in urethral resistance leading to an increase invoiding pressure and wall tension [399,420-424]. Theincreased wall tension or voiding pressure result in anincrease in bladder mass (and wall thickness)[177,178,181,408,425,426]. The increase in bladderwall thickness results in cyclical periods of ischaemia-reperfusion during bladder contraction and emptying[422-4]. The relationships between intravesical pressu-re and blood flow, and intravesical pressure and detru-sor pO2 during and following micturition contractionsare presented in figure 30. These studies have clearlydemonstrated that in obstructed pigs there is a directcorrelation between increased intravesical pressure(wall tension) and decreased blood flow. During thereduced blood flow, there is clearly a substantialdecrease in detrusor pO2. Although blood flow is resto-

red rapidly after the end of the bladder contraction,detrusor pO2 returns toward normal levels much moreslowly. These same studies demonstrated that overacti-ve bladder contractions result in a similar phasic reduc-tions of blood flow, and pO2. Thus, obstructed pigs thatshow detrusor overactivity have a greater frequency ofischaemic periods than obstructed pigs that show nooveractivity.

The relationship between ischaemia and obstruction issupported by a recent study by Schr�der et al. whodemonstrated in rabbits that blood flow to the detrusorsmooth muscle was reduced in proportion to the levelof decompensation [427]. Interestingly, blood flow tothe mucosa was not affected by obstruction (figure 31).Similar results have been reported [428]. Duringischaemic periods there is a release of intracellularCa2+ from the SR which can stimulate Ca2+-activatedhydrolytic enzymes such as calpain (calcium activatedprotease) and phospholipase A2 (Ca2+ activated phos-pholipase) [429-431]. During the reperfusion period,free radicals can be formed which stimulate lipid per-oxidation. The increase in the activities of these degra-dative enzymes result in damage to specific membraneelements within the detrusor.

Ischaemia will damage not only smooth muscle cellsbut also the intrinsic neurones in the bladder wall.

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Table 8 : Effect of Partial Outlet Obstruction on Bladder Smooth Muscle Metabolism

Control ObstructedSmooth Muscle Smooth Muscle

Glucose metabolism

Lactic acid formation (:mol / g / h) 1.35 ± 0.05 1.84 ± 0.10**

CO2 formation (:mol / g / h) 0.26 ± 0.01 0.17 ± 0.01**

High energy phosphate content

Creatine phosphate (nmol / mg protein) 18.2 ± 2.4 7.5 ± 1.0**

Adenosine triphosphate (nmol / mg protein) 8.2 ± 2.0 7.2 ± 1.3

Adenosine diphosphate (nmol / mg protein) 4.0 ± 0.3 3.2 ± 0.6

Adenosine monophosphate (nmol / mg protein) 4.0 ± 0.3 3.5 ± 0.6

Enzyme activity

Citrate synthase a (nmol / mg protein) / min) 286 ± 25 140 ± 28**

Malate dehydrogenasea (nmol / mg protein) / min) 464 ± 43 280 ± 30

NCCRa (nmol / mg protein) / min) 406 ± 24 394 ± 13

Cytochrome oxidasea (nmol / mg protein) / min) 163 ± 20 131 ± 20

Creatine kinase (nmol / mg protein) / min) 582 ± 50 505 ± 30**

Adenylate kinase (nmol / mg protein) / min) 50 ± 5 61 ± 10**

Phosphoribosyl transferase (nmol / mg protein) / min) 33.7 ± 2.7 29.0 ± 4**

Myosin ATPase (:mol / mg protein / min) 1.61 ± 0.16 1.63 ± 0.33

Hexokinase (nmol / mg protein) / min) 11.7 ± 1.6 10.1 ± 1.0

NCCR - NADH-cytochrome c reductase. a - Activity in partially purified mitochondrial preparation; All data are given as means± SEM; **significantly different from smooth muscle (P<0.05).

Indeed evidence strongly suggests that neurones aremore susceptible to ischaemic damage than the detrusorsmooth muscle and also that such damage is more like-ly to be irreversible. [189,190,200,201]. Ischaemic neu-ronal damage can be reduced if the tissues are treatedwith L-type Ca channel blockers, or exposed to ischae-mic conditions in Ca-free solutions, or in the presenceof free radical scavengers. The protective effect of L-type Ca channel blockers and free radical scavengersstrongly suggests that free radicals produced by, andreleased from, the smooth muscle cells may be partial-ly responsible for the neuronal damage, since L-type Cachannels are predominantly on the smooth muscle cells,and blockers do not prevent activation of intrinsicnerves and release of neurotransmitters [253]. Neuronaldamage is commonly seen in obstructed unstable blad-ders and may be an important factor in causing thechanges in smooth muscle properties associated withoveractivity [1].

Free radicals produced in ischaemia will, through acombination of damage to neurones and to the smooth

muscle itself, also begin and promote the process ofdecompensation. In man, there is clear clinical supportfor the concept that the rate of bladder deterioration inthe presence of obstruction is significantly greater ifinvoluntary bladder contractions (hyperreflexia) arepresent. This concept is clearly supported by experi-mental evidence in animal models. In both the guineapig and rat models of partial outlet obstruction, obs-tructed animals that show hyperreflexia clearly show agreater level of dysfunction than animals obstructed forthe same period of time that do not show hyperreflexia[432,433].

Repetitive stimulation has been used to simulate detrusoroveractivity using in vitro methodologies. These studiesutilized normal rabbit bladder detrusor strips mounted inindividual isolated baths. The effect of repetitive stimu-lation using 32 Hz field stimulation on the contractileresponses of the strips to field stimulation, carbachol,and KCl was determined. The results of these studiesdemonstrated that isolated strips that were subjected torepetitive stimulation showed a significantly greater rateof contractile failure, and show a significantly reducedability to recover contractile function during re-oxygena-tion in the presence of glucose. Hypoxia exaggerated thedestructive effects of repetitive stimulation [434,435]. Inaddition, tissues were analysed for MDA (malondialde-hyde: a peroxidation product of free radical damage).There was a clear correlation between the severity of thecontractile dysfunctions, the duration of the repetitive sti-mulation, and the concentration of MDA [434,435].Additional studies demonstrated that the degree of dys-function and MDA generation was directly related to thebath concentration of Ca2+. Increasing calcium in thebath increased the rate and magnitude of the contractiledysfunctions and increased MDA concentration, whereaslow calcium, EGTA, and the presence of Ca2+ blockingagents in the bath protected the contractile responses.These studies provide additional support that the detri-mental effects of overactive bladder contractions aredirectly related to both the presence of ischaemia andCa2+ cycling.

2. AGING

¥ RELATIONSHIP BETWEEN CELLULAR METABOLISM AND

AGING

Reduced energy supply (rate of ATP generation) causesa progressive decrease in the tonic response of the ure-thra, the loss of aÓ-adrenergic tonic tension, and adecreased urethral pressure, contributing to incontinen-ce in women. In men, the decreased ability of the blad-der to generate and sustain a micturition contractionwould result in decreased flow rate, increased time ofmicturition, and increased residual volume. Thesesymptoms are well documented to be associated with

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Figure 31 : Effect of partial outlet obstruction on bloodflow to the detrusor smooth muscle and mucosa. 15 NewZealand White rabbits received a partial outlet obstructionby standard methods. After 4 weeks the rabbits were anaes-thetized and blood flow to the muscle and mucosa weredetermined by a standardized fluorescent microsphere tech-nique. A section of each detrusor was used for in vitrocontractility studies. Contractile responses to field stimula-tion (FS), carbachol, ATP, and KCl were determined andutilized in addition to bladder weight to determine the levelof decompensation present. Each bar is the mean of 5control, 5 compensated, 5 intermediate-decompensated ,and 5 decompensated rabbit bladders. * = significantly dif-ferent from control, p < 0.05.

aging, even in the absence of any obstruction. Thisincreased rate of ÒfatigueÓ in association with aging hasbeen proposed by Lin et al who demonstrated that inageing rats there was a progressive decrease in thecontractile responses to repetitive autonomic stimula-tion (increase in fatigue) which was directly associatedwith both a decrease in the metabolic energy availableto support the contractile responses, and a decrease inmitochondrial enzyme activities [436,437]. Thus, inassociation with ageing, there is a progressive reductionin the rate of high energy phosphate generation (duringperiods of stimulation) that significantly increases therate of fatigue of the bladder smooth muscle. Anotherstudy demonstrated a significantly reduced ability ofisolated whole bladders from aged rabbits (as comparedto those from young rabbits) to generate pressure andempty efficiently [438]. The rate of power generation,the percent emptying, and the amount of work perfor-med by bladders from old rabbits were significantlyless than bladders from young rabbits. In addition, therewas a marked reduction in the ability of the bladdersisolated from old rabbits to respond to an increase inoutlet resistance. These studies support the theory thatageing is associated with a progressive reduction in theability of the bladder to generate metabolic energy byoxidative phosphorylation, and thus sustain increasedtension. Decreased mitochondrial oxidative function inassociation with aging have been reported in a numberof different biological systems.

a) Physical properties of the Bladder in aging

In addition to the metabolic changes, the volume andthe elasticity of the bladder can change in aged humansand experimental animals.

Clinically, the frequency of micturition and the voidedvolume of urine, are known to increase, and there is areduced ability to suppress the bladder once fullnesshas been noted. These signs and symptoms are accom-panied by urodynamic changes including an increasedresidual volume, and a reduction in the maximumsqueeze pressure generated by the urethra [439]. Theincreased residual urine and the lower uroflow ratefound in the elderly may also reflect changes in thebladder muscle leading to less efficient and unsustainedcontractions [440,441]. In animal experiments it hasbeen suggested that the volume of the bladder was simi-lar in young and old rats, but that micturition occurredat higher pressures in the older animals [442,443] .However that is not universally accepted, and Hotta etal (1995) found that the bladders of old animals can beconsiderably larger than those of young animals, andthe urine can be accommodated at low pressure [444].

b) Afferent and Efferent Nerve Function inAging

Gilpin et al [445] found that the amount of nerve per

mm2 of muscle decreases with age and occurs to thesame extent in males and females. Warburton and San-ter [446] presented evidence which suggested a dimi-nution in the sympathetic control of the urinary tract inaged rats; they also found however that the pattern anddensity of CGRP-containing afferent nerve terminals inthe bladder were unchanged in old age. Thus it may bethat old age causes a reduction in the innervation densi-ty of the bladder, just as it does for skeletal muscle[447].

From a functional viewpoint, micturition threshold(pressure) is reported as being unchanged [444]. Howe-ver it was found that the afferents were less sensitive tovolume, while retaining their sensitivity to pressure.Corresponding to this finding in rat bladders, Collas etal [448] also found a decrease in bladder sensation inassociation with aging in female patients with lowerurinary symptoms. They found that maximum bladdercapacity fell in the eighth and ninth decades; whereasthe bladder capacity at first desire to void rose progres-sively in association with age, which is consistent withthe rat data.

The responsiveness of vesical smooth muscle to neuro-transmitters and other chemicals may or may not chan-ge in old age. To some extent this is dependent on whatone considers ÔoldÕ; many of the changes seen in rats at>26 months of age are not seen below the age of 20weeks. Toyoshima et al [449] found that the responsi-veness to acetylcholine is reduced by aging, and sug-gested that the increase in intravesical pressure thatoccurs during stimulation with cholinergic agonistsinvolves the participation of cholinergic M-2 receptors.Munro and Wendt [450] also found that the maximalforce generated by the aged muscle strips decreased andthat the maximal velocity of shortening was signifi-cantly lower in strips from aged animals as comparedwith those from young adults. However others havefound that there was no significant age-related differen-ce in the response to cholinergic ands other contractilestimuli [451-453]. On the other hand, there are nume-rous reports of aging-related changes of the detrusorresponse to adrenergic stimulation. Most studies sho-wed that detrusor contractile responses to a-adrenergicstimulation increased in old male and female rats [453-5]. The detrusor response to b-adrenergic stimulation isreduced in old male rats [453,454] along with a reduc-tion in the density of b adrenergic receptors and adecreased cyclic AMP (cAMP) production in responseto b adrenergic stimulation. The combination of anincrease in the a-adrenergic excitatory responses anddecreased b adrenergic inhibitory responses results in anet contracting effect of noradrenaline on the agedbladders in contrast to the relaxing effect of noradrena-line in the young bladders. When noradrenaline is relea-sed by increased sympathetic activity during bladderfilling [5] the enhanced adrenergic contractile response

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in the aged bladders might reduce functional storagecapacity and thereby contribute to urinary frequencyand urgency in the older persons.

In in vivo experiments, Hotta et al [444] found that boththe active and the passive pressure-volume relation-ships were shifted to the right in the elderly animals,and that aging was associated with a reduction in themaximal pressure generated during pelvic nerve stimu-lation for 10 seconds at 20 /second, using square wavepulses of 10V and 1.0 msec. Also the change in intra-vesical pressure induced by supramaximal nerve stimu-lation was less in aged animals. Several studies haveshown that the frequency-response curve for electrical-ly evoked bladder contractions was similar in youngand old rat bladders [442] indicating that the release ofneurotransmitters from pre-synaptic nerve endings wasnot altered during aging.

3. OUTLET OBSTRUCTION

The development of a spinal micturition reflex with sub-sequent detrusor overactivity occurs in conditions otherthan spinal cord injury. Bladder outlet obstruction in ratscauses enhancement of a spinal reflex [456,457]. Withinthe spinal cord obstruction stimulates an increasedexpression of GAP-43 which has been associated withaxonal sprouting following injury [458]. These observa-tions suggest an enhancement or de novo development ofnew spinal circuits following obstruction. Similar to spi-nal cord injury, obstruction causes hypertrophy of blad-der afferent and efferent neurons [391,392]. Conversely,relief of obstruction is associated with the reduction ofurinary frequency and reversal of these neural changes[458,459]. In animals that fail to revert to a normal voi-ding pattern after relief of obstruction, this neuroplastici-ty persists [459]. Nevertheless, these findings are notmutually exclusive of changes in the bladder smoothmuscle that are also likely to participate in the develop-ment of the overactive bladder [460].

Bladder outlet obstruction also appears to initiate themorphological and electrophysiological afferent plasti-city via a mechanism involving NGF (Fig.32). NGF isresponsible for the growth and maintenance of sympa-thetic and sensory neurons and has been shown to beresponsible for neuronal regrowth after injury. NGFcontent is increased in obstructed bladders in animalsand in humans [461]. This increase in NGF content pre-cedes the enlargement of bladder neurons and the deve-lopmental of urinary frequency [375,461]. Moreover,blockade of NGF action using autoantibodies preventsthe neural plasticity and urinary frequency followingobstruction [461]. In animals in whom urinary frequen-cy persists after relief of obstruction, NGF remains ele-vated in the bladder. Although mechanical stretch ofcultured bladder smooth muscle induces NGF expres-sion, other insults to the bladder such as denervation

and ischemia are capable of inducing NGF. These fin-dings suggest a cause and effect relationship betweenNGF-mediated changes in bladder afferents and anenhanced spinal micturition reflex and urinary frequen-cy associated with obstruction. Of interest, a-adreno-ceptor and NK1 antagonists and oxytocin, administeredintrathecally, can reduce unstable bladder contractionsin obstructed rats [462]. These findings support thenotion that changes in the spinal cord, possibly relatedto alterations in the processing of afferent input contri-bute to the bladder dysfunction after outlet obstruction.

4. INFLAMMATION

Because NGF plays such a prominent role in the deve-lopment and function of afferent neurons, it is no sur-prise that inflammation of the urinary bladder is accom-panied by neuroplasticity in sensory nerves supplyingthe bladder [95]. Repeated inflammatory stimuli elicitsenlargement of bladder DRG neurons [463]. Followinginflammation a reduction in threshold for bladder affe-rents occurs [595]. Likewise, it has been shown thatintravesical NGF can lower thresholds for bladder affe-rents. Plasticity within the spinal cord may also occur.Following chemical or mechanical inflammation of theurinary bladder, increased expression of the early-immediate gene, C-fos, has been detected within thelumbosacral spinal cord [465-467] and increasedexpression of nitric oxide synthase occurs in bladderafferent neurons [596].

Although inflammation of the urinary bladder elevatesNGF content, this rise is short lived [468]. Bladder ove-ractivity induced by inflammation can be inhibited by afusion protein that prevents interaction between NGFand its tyrosine-(trk-A) receptor [469]. Hence, the neu-roplasticity and the potential involvement of NGF ininflammatory conditions resemble that of obstruction.Other substances including neurotrophins, prostaglan-dins, and tachykinins may also contribute to changes inafferent excitability [6].

Models of bladder overactivity involving primarylesions within the CNS have been developed in severalspecies, and are relevant to spinal cord injury, denerva-tion, ParkinsonÕs disease and multiple sclerosis [5,470].In addition, the spontaneously hypertensive rat (SHR)has provided a useful genetic model for bladder ove-ractivity [471]. One common theme behind all of thesemodels is that acute changes in smooth muscle can eli-cit long-term changes in nerves. Investigators areaccustomed to examining short-term effects. However,there is now a greater appreciation that long-termevents involving growth factors leads to plasticity in

II. CENTRAL MODELS OF OAB

135

neural pathways with implications for disorders of mic-turition. Neurotransmitters, prostaglandins and growthfactors, such as Nerve Growth Factor (NGF), are sub-stances that provide mechanisms for communicationbetween muscle and nerve. Disturbances in thesemechanisms can cause bladder overactivity due to alte-rations in either bladder smooth muscle or autonomicreflex pathways. This bladder overactivity can, in turn,lead to urge incontinence.

Cystometry and urinary frequency are commonly usedto define bladder overactivity. Moreover, these measu-rements can monitor responses to drugs or other thera-pies. A multidisciplinary approach incorporating one ormore biochemical, molecular, pharmacological, phy-siological and behavioral methods can provide insightinto the pathogenesis of detrusor overactivity. (Table 9)

1. SPINAL CORD INJURY

Damage to the spinal cord cephalad to the sacral levelresults in bladder overactivity [472]. Acute spinal cordinjury disrupts normal supraspinal circuits that controlurine storage and release. Following days to weeks ofurinary retention, hyperactive voiding develops. Elec-trophysiological data reveal that this detrusor hyperre-flexia is mediated by a spinal micturition reflex thatemerges in response to a reorganization of synapticconnections in the spinal cord [9,44,275,471]. In addi-tion, bladder afferents that are normally unresponsiveto low intravesical pressures become more mechano-sensitive leading to the development of detrusor ove-ractivity. Normal micturition is associated with a spino-bulbo-spinal reflex mediated by lightly-myelinated A-dafferents [5,9]. These fibers represent only 20% of blad-

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Figure 32 : Results from a wide array of pharmacological, molecular and electrophysiologic experiments suggests a similarunderlying mechanisms for changes in innervation that in turn could potentially lead to afferent overactivity in variety ofmodels for overactive bladder (OAB). Models of bladder inflammation, outlet obstruction, spinal cord injury, and the sponta-neous hypertensive rat (SHR) all exhibit urinary frequency, reduction in volume threshold to micturition, and intermicturitioncontractions (unstable) similar to OAB in humans. All of these models are associated with elevated levels of NGF as are someclinical conditions as interstitial cystitis and obstruction. Neuronal changes occur in the dorsal root ganglia (DRG) and dor-sal horn (DH) of the spinal cord. Afferent project along a lateral collateral pathway (LCP) to the sacral parasympatheticnucleus (SPN). In the DRG hypertrophy of neurons, lowered threshold for activation of neurons giving rise to C-fibers, andchanges in neuropeptide (NP) content are found. In the DH, up-regulation of growth associated protein 43 (GAP43), neuro-nal nitric oxide synthase (nNOS), and the early immediate gene product cfos occur. Moreover, antagonists to neurokinin1(NK1) an a1 adrenoceptors administered intrathecally inhibit OAB with these disorders. Following chemical irritation andobstruction, blockade of NGF antivity with trkA antibody or autoimmunity to NGF block neuronal plasticity and OAG.

der afferents in some species. Compared to A-d fibers,the more prevalent unmyelinated C-fibers are relativelyinsensitive to gradual distention of the urinary bladder,at least in the cat [29]. Most C-fibers in this speciesremain silent during normal filling of the bladderalthough in the rat some studies indicate that C-fiberscan fire at low pressures [26]; whereas other studies[24] showed firing at higher intravesical pressures ofapproximately 30 mm Hg. After spinal cord injury, acapsaicin-sensitive C-fiber-mediated spinal reflexdevelops (Fig 33). These C-fiber afferents are thoughtto play a role in the development of bladder overactivi-ty after spinal cord injury. Capsaicin sensitive C-fibershave also been implicated in detrusor overactivity fol-lowing upper motoneuron diseases such as multiplesclerosis and ParkinsonÕs disease [473-475].

Insight into the mechanism underlying the increasedmechano-sensitivity of C-fibers after spinal cord injuryhas been gained by examining the dorsal root ganglion(DRG) cells supplying the bladder. Plasticity in theseafferents is manifested by enlargement of these cells[474] and increased electrical excitability [475]. A shiftin expression of sodium (Na+) channels from a highthreshold tetrodotoxin (TTX)-resistant type to a lowthreshold TTX sensitive type occurs after spinal cordinjury.

Plasticity in bladder afferents after spinal cord injurymay involve the retrograde transport of substances fromeither the spinal cord or bladder to the DRG neuron.Bladder DRG neurons are responsive to a variety ofneurotrophins, especially NGF which has been associa-ted with hypertrophy of bladder DRG cells in a varietyof conditions (see Obstruction, Inflammation, Sponta-neously Hypertensive Rat). Exposure of cultured DRGneurons to exogenous NGF promotes expression of

TTX sensitive channels expressed following spinalcord injury [478]. Moreover, DRG from mice overex-pressing NGF exhibit predominately TTX-S Na cur-rents. Other trophic substances such as basic fibroblastgrowth factor (bFGF), brain-derived neurotrophic fac-tor (BDNF), glial derived neurotrophic factor(GNDF),and neurotrophins-3-4 (NT-3-4) may triggermorphological and electrophysiological alterations inDRG cells after spinal cord injury. GDNF may be espe-cially important because a small population of DRGneurons giving rise to C fibers are non-reponsive toNGF, but respond to GDNF [478]. It is worth notingthat the response of other neurogenic disorders associa-ted with urge incontinence respond to intravesical cap-saicin therapy suggesting that plasticity in C-fiber affe-rents could form the neurogenic basis for bladder ove-ractivity [474,475,479]. The emergence of a spinalreflex circuit activated by C-fiber bladder afferentsrepresents a positive feedback mechanism (Fig. 34) thatmay be unresponsive to voluntary control by higherbrain centers and thereby be able to trigger involuntaryvoiding.

2. SPONTANEOUSLY HYPERTENSIVE RAT (SHR)

The spontaneously hypertensive rat (SHR) voids morefrequently at lower volumes than genetic Wistar-Kyoto(WKY) controls and has overactive bladder contrac-tions even though they produce equal amounts andconcentration of urine. Urinary frequency, urgency withor without urge incontinence and involuntary bladdercontractions characterize the overactive bladder (OAB)[371]. Thus the SHR may be a useful model to studyeither the treatment or pathophysiology of OAB. Thereason for OAB in the SHR is unclear. Bladder smoothmuscle from the SHR produces more nerve growth fac-tor (NGF) than WKY [480-482]. The bladder alsodemonstrates a hypernoradrenergic innervation andincreased responsiveness to adrenergic agonists[480,483]. However, changes in sympathetic nerves orsmooth muscle responsiveness do not fully explain theOAB. The only significant in vitro finding is an enhan-ced b- adrenergic mediated relaxation. In vitro studiesshow equivalent responses to electrical stimulation,potassium chloride, cholinergic and purinergic agonistsin SHR and WKY. SHR and WKY are similar in termsof histology and bladder compliance during filling.These findings do not implicate changes in smoothmuscle or connective tissue. Furthermore, chemicalsympathectomy with elimination of noradrenergicnerves by 6-hydroxydopamine or prevention of nora-drenaline release with guanethidine fails to abolishOAB.

It can be inferred from several lines of evidence thatafferents may be altered in the SHR. Published data

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Table 9 : Legend: NF = neurofilament, IB4= lectin SP=substance P CGRP=calcitonin gene related peptide, trkA-tyrosine kinase A , VR1- vanilloid receptor 1.

60% of bladder afferents are C-fibers

Peptidergic Non-Peptide

NF - NF -

80-90% bladder C fibers 10-20% bladder C fibers

IB4 ÐSP/CGRP IB4/FRAP +

Trk A-NGF Trk B-BNDF GDNF

VR1 ?VR1/ P2X3

Laminae I/II Inner laminae II

Projection 2nd order to 2nd order interneurons in brainstem/thalamus spinal cord

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Figure 33 : Neuropharmacology within dorsal horn of sacral spinal cord. Afferents projecting to the dorsal horn are of twotypes Ad and C. These afferents release glutamate, substance P (SP), nitric oxide (NO), or pituitary adenlyate cyclase (PACAP)and adenosine triphosphate (ATP) among other neurotransmitters. Glutamate, SP, and PCAP are excitatory although PACAPprobably modulatory. Glutamate acts at NMDA and AMPA ionotropic or metabotropic glutamate receptors. SP acts at neuro-kinin Ð (NK-1) receptors. Afferents from the lower urinary tract can synapse on second order neurons can either project ros-trally to the periaqueductal gray (PAG) or on interneurons, most of which also contain glutamate. Modulatory influences onsecond order neurons derives from inhibitory GABAminergic interneurons (acting on predominately GABAB receptors) ordescending corticotropic releasing factor (CRF), oxytocinergic (OXY), or serotonergic (5-HT2C, 5-HT1A) inhibitory neurons.Descending noradrenergic neurons acting at a1D or a2C receptors can exert either excitatory or inhibitory influences on mic-tuition.

Figure 34 : Following inflammation andcystitis a rise in TTX-R Na currents ismeasured in bladder DRG. After spinalcord injury or in SHR increased TTX-SNa currents is seen. A reduction in slowinward delayed rectifier K currents mayreduce hyperpolarization and causeincreased neuronal excitability. Thus,elevated NGF may act to influence asubunit exression which affects the exci-tability of neurons. This excitability maybe manifested in a variety of ways such asa reduction in threshold, spontaneous orburst firing of afferents. Such an increa-se in afferent excitability could lead to areduction in volume threshold for mictu-rition in OAB conditions.

reveals that DRG neurons are enlarged in SHR compa-red to WKY. Intrathecal administration of the a antago-nist doxazosin, which may modify afferent input fromthe bladder, inhibits OAB in SHR. Doxazosin has noeffect on bladder function in WKY. One possibility isthat NGF produced in SHR influences the electrophy-siologic properties of L6/S1 dorsal root ganglion(DRG) cells supplying the SHR bladder differ fromother strains with regard to Na currents. These Na cur-rents regulate the excitability of afferents. Moreover,intrathecal antisense (AS) oligonucleotides (AG)against the gene (SCN10A) encoding for Nav1.8, atetrodotoxin resistant (TTX-R) Na channel isoformraises the volume threshold for micturition and reducesunstable bladder contractions in SHR [484]. Thereforeit seems reasonable to explore the differences in blad-der afferents and CNS pathways in the SHR comparedto WKY as a basis for OAB. The precise mechanism(s)giving rise to OAB in the SHR may not be identical tothose manifested in clinical entities such as idiopathicdetrusor overactivity, interstitial cystitis, and bladderoutlet obstruction or spinal cord injury. However expe-rimental manipulations including urethral ligation andchemical cystitis do not mimic the spectrum of changesseen clinically with interstitial cystitis or benign prosta-tic hyperplasia (BPH). Nevertheless these models haveprovided insight into the potential mechanisms leadingto OAB. The SHR offers substantial advantages as amodel for OAB. For example, animal welfare concernsare less an issue in the SHR than with surgical or che-mical manipulations. Likewise, unraveling the molecu-lar events involved in the SHR may uncover novelapproaches to therapy and provide insight into themechanisms whereby these symptoms arise.

BPH and hypertension (HTN) often coexist in the agingpopulation. For unknown reasons the prevalence of uri-nary symptoms is greater in hypertensive men andwomen [485-490]. Recent work presented at the 5thInternational Consultation on BPH suggests that HTNis the major risk factor for symptoms documented by anInternational Prostate Symptom Score (IPSS)(P. Boylepersonal communication). A subset of hypertensivepatients exhibits traits indicative of an overactive auto-nomic nervous system. In hypertensive, but not in nor-motensive controls, bladder distension evokes a briskparasympathetic response of the baroreceptor/heart ratereflex [491]. An elevated heart rate response to stresshas been seen in normotensive offspring of hypertensi-ve parents [492]. Cross breeding SHR and normotensi-ve WKY rats creates a F1 generation in which bloodpressure correlates with urinary frequency [493].However, it is unlikely that HTN per se causes a chan-ge resulting in OAB in the SHR. Urinary frequency ispresent at age 3 wks in SHR at which time the animalsare normotensive. Moreover, adequate control of HTN

in patients does not preclude OAB symptoms. This cir-cumstantial evidence suggests that a subtle enhance-ment of autonomic activity may lead to HTN and pre-dispose to OAB. Whether a difference exists in autono-mic afferents, efferents or smooth muscle between SHRand WKY and whether this difference explains alteredvoiding behavior is unclear.

¥ PLASTICITY IN NA CHANNEL ISOFORMS

How could NGF produced by a target tissue and thenretrogradely transported to the DRG affect target organfunction? Recent data suggest that membrane conduc-tance and thus excitability of DRG neurons are alteredin models associated with increased access to NGF.This altered conductance is postulated to result fromchanges in the structure or combination of protein subu-nits of Na and K channels in the cell membrane. Achange in Na channel isoform appears sufficient tochange the properties of afferents. NGF is known tolower the threshold for firing of bladder (hypotheticallower volume threshold for voiding)and induce sponta-neous and burst firing (hypothetical overactive contrac-tions, urgency) [95]. Examples of afferent plasticityinduced by NGF include inflammation or axotomy,which trigger spontaneous firing of sensory neurons[494] (Table 10). The site of abnormal ectopic firinghas been determined to be the DRG and appears to bedue to changes in the isoform of the voltage gated Nachannels, causing spontaneous ectopic discharges. Vol-tage gated Na+ channels generate currents during theupstroke of the nerve action potential and are involvedin the propagation of the nerve impulse. Tetrodotoxin(TTX) is a neurotoxin produced by puffer fish, and itbinds to and inactivates voltage gated Na+ channels.

The Na+ channel is a heterotrimer of composed of a,b1, b2 subunits (Fig 35). Subunit composition conferselectrical properties and TTX susceptibility. Distinctgenes code for the voltage-gated Na+ channel subunitsthat control membrane excitability of DRG neurons.These genes code for a and b subunits that assemble toform channel isoforms that have been distinguished onthe basis of structure, biophysical properties, and sensi-tivity to tetrodotoxin (TTX) [495]. The a subunit formsthe pore of the Na channel. The b subunit serves as anadhesion molecule to localize the channel [597]. Lossof the b subunit may prevent TTX binding implyingthat b subunits can also influence function. The propor-tion and characteristics of Na+ currents determine therange of firing patterns and functional properties inDRG neurons.. Na channel isoforms, like those for Kchannels, are distinct in terms of developmental andregional expression. Most voltage-gated Na+ channelsexhibit rapid inactivation kinetics and sensitivity tonanomolar concentrations of TTX. These Na+ channelsare termed TTX-sensitive (TTX-S). Small sensory neu-

139

rons in the L6/S1 DRG show two types of Na+ cur-rents, a rapidly inactivating TTX-S INa and a slowlyinactivating TTX-resistant (TTX-R) INa.

As neurons switch from a quiescent state to high fre-quency firing, such as with reawakening of C-fibers,they use their Na channels differently. But does DRGemploy a different repertoire of Na channels? There isextensive evidence to suggest that Na+ channel subu-nits change in response to environmental conditionsand changes in access to NGF. Two strategies are usedin general to assess Na channel isoforms. First, studiescan assess mRNA or protein production using in situhybridization or immunohistochemistry for Na channelisoforms. Second, biophysical properties of cells can beassessed using patch clamp techniques. Using thesemethods, it has been discovered that Na channel iso-forms in cells can vary by species, with disease and fol-lowing acquired conditions such as inflammation andaxotomy.

Does plasticity in Na channel currents occur in neuronssupplying the bladder? 61% of sensory nerves innerva-ting the rat bladder are unmyelinated C-fibers based onconduction velocities of single dorsal root fibers [31].The remaining nerves are lightly myelinated Ad fibers.61% of bladder afferents respond to distension of thebladder (Shea et al 2000). 31% fail to respond to anymechanical or chemical stimuli. At least 60% of blad-der DRG neurons are capsaicin-sensitive (VR-1), 95%of which give rise to C-fibers [496]. The TTX-R Nacurrents are present in SP/CGRP immunoreactive, trkAexpressing, small DRG giving rise to C-fibers sup-plying the bladder [496,497]. Greater than 85% of Nacurrents in small diameter (C-fiber) DRG that accoun-ted for 70% of bladder labeled cells exhibit predomina-tely TTX-R currents. These cells demonstrate high thre-sholds and long duration action potentials and likelycontribute to the higher thresholds for C-fibers. Theremaining 30% of DRG neurons giving rise to Ad fibers

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Figure 35 : Excitability of nerves is (action potential generation ) attributable in part to Na currents. The Na channel is a hete-rotrimer composed of an a subunit forming the pore and two b subunits that anchor the channel to a membrane. Eight iso-forms for the Na channel have been identified based on changes in the a subunit. Electrophysiologic properties of neuron lin-ked to subunit expression which varies during development and with disease.

exhibit predominately TTX-S Na currents. These cellsshow low thresholds and short duration action poten-tials. After spinal cord injury, obstruction, and cystitis,an enhancement of spinal reflexes has been confirmedby abolishment with capsaicin and a positive ice-watertest clinically [306,598]. Plasticity occurs in TTX-S andTTX-R Na+ channel currents in DRG neurons after spi-nal cord injury. With spinal cord injury 60% of disso-ciated bladder DRG neurons exhibit TTX-S Na currentscompared to 30% in intact rats [498]. IA K currents arealso postulated to be reduced causing lack of membra-ne relaxation. These two changes lead to enhancedexcitability of C-fibers coinciding with development ofa spinal micturition reflex. With cyclophosphamidecystitis, slowly inactivating IA currents in TTX-R neu-

rons are reduced possibly leading to OAB [498]. It hasbeen speculated that the cellular basis for this plasticityis a change in K currents.

¥ CLINICAL EVIDENCE FOR ROLE OF Na CHANNELS IN OAB

Observations from use of the local anesthetic and non-selective Na channel blocker, lidocaine, suggests that Nachannels can be manipulated to reduce OAB in a varietyof conditions including BPH. Intravesical lidocaine atlow doses blocks sensory nerve transmission from thehuman bladder. The effect of intravesical lidocaine maybe limited to C-fibers giving rise to pathologic reflexessuch as the ice water test [499]. Intravesical lidocaineincreases bladder capacity and reduces involuntarycontractions. In a small, uncontrolled study of elderlypatients with detrusor instability, the oral Na channelblocker, mexiletine, but not intravenous lidocaine,improved or cured urge incontinence [500]. The effectsof these agents vary with disease. This could indicatealterations in Na channel expression by nerves, diffe-rences in the degree of innervation or access to drug. Inchildren with neurogenic bladders due to myelodyspla-sia, Lapointe and colleagues [501] found that intravesicallidocaine decreased involuntary contractions in 58% ofpatients but increased capacity in 71% of patients bynearly 60%. Lidocaine increases bladder capacity to thegreatest degree in spinal cord injured patients (230%)compared to supraspinal neurologic conditions and BPH[502]. A reduction in involuntary detrusor contractions isonly seen in the former conditions but not BPH. Incontrast, Reuther and colleagues [503] found that elimi-nation of detrusor overactivity after intravesical lidocai-ne was predictive of cure of symptoms after transurethralresection of the prostate. Intravesical lidocaine can evenworsen detrusor overactivity in some BPH patients[504]. It is possible that the action of intravesical lido-caine is primarily limited to suburothelial nerves. Thiswould be consistent with observations in other studiesthat periprostatic or subtrigonal injection of lidocaine eli-minates urgency and frequency associated with BPH[505]). Regardless, bladder or prostatic afferents expres-

sing Na channels may be involved in irritative voidingalthough not the cause for involuntary contractions. Dif-ferences between these small studies might be attributedto ischemia or denervation. These conditions couldnecessitate a higher dose of lidocaine to eliminate invo-luntary contractions in BPH patients. In obstructed rab-bits, intravesical lidocaine but not anticholinergics,potassium channel openers or Ca2+ channel antagonistsinhibited hyperreflexia [506]. Lidocaine eliminates painassociated with intravesical capsaicin indicating its abili-ty to inhibit vanilloid 1 receptor (VR-1) expressing affe-rents probably located beneath the urothelium. Incontrast, intravesical tetrodotoxin fails to block intravesi-cal capsaicin-induced abdominal licking in rats implyingthat VR-1 expressing bladder afferents are TTX-R [507].However, targeting Na channels expressed by suburothe-lial nerves may not be as effective as blocking thoseexpressed by spinal nerves. Intrathecal lidocaine andespecially bupivacaine eradicate urinary urgency andincrease bladder capacity [508]. Therefore manipulationof Na channels expressed by spinal nerves or afferents inDRG could influence micturition. (Table 10).

3. MODELS OF PARKINSONÕS DISEASE WITH

OAB

Chemical or surgical lesioning of dopamine containingneurons in the basal ganglia in the rat causes changes inmotor behaviors reminiscent of ParkinsonÕs disease.CMGs in this animal demonstrate detrusor overactivity.Projections of dopamine containing fibers from the sub-stantia nigra have opposing effects on the bladder. Acti-vation of D2 receptors is excitatory to micturition.Conversely, release of dopamine from rostral projec-tions results in activation of D1 receptors which ele-vates the threshold to micturition. This latter effect hasbeen postulated to result from activation of D1 recep-tors on GABAminergic neurons which mediate inhibi-tion of neurons involved in micturition. In a model ofParkinsonÕs disease induced by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), investigators foundthat D1 function is reduced and D2 activity is enhancedwith a net excitatory effect on micturition reflexesmanifested as a reduced volume threshold for voidingand unstable bladder contractions. An animal model forParkinsonÕs disease has been developed in monkeys byadministering a neurotoxin (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP]) that destroys dopa-mine neurons [599]. Animals treated with MPTP showmotor symptoms typical of ParkinsonÕs disease and alsohave an overactive bladder. In these monkeys, stimula-tion of D-1 receptors with SKF38393 suppressed thedetrusor hyperreflexia, whereas a nonselective dopami-ne receptor agonist (apomorphine) or a D-2 receptoragonist (quinpirole) reduced the bladder volume thre-shold.

141

4. STROKE

Ligation of the middle cerebral artery of the rat createsbrain ishemia and cerebral infarction. This animal exhi-bits an OAB characterized by intermicturition contrac-tions on CMGs in conscious rats which supports thenotion of tonic cortical inhibition of bladder function[354,509]. This effect was eliminated by administeringan NMDA glutamatergic receptor antagonist, indicatingthat glutamatergic excitatory transmission is involvedin the OAB [354]. Examination of other pharmacologicresponses to intracerebroventricular drugs such asGABAminergic and dopaminergic agents drugs revea-led an decreased GABA mediated inhibition of micturi-tion and dopaminergic influences and glutamate trans-mission. Thus cerebral infarction causes plasticity ininhibitory neurons resulting in OAB.

5. OAB IN CLOMIPRAMINE MODEL AND

DEPRESSION

Wolfe et al, [510] suggested that depression, anxiety,feeding disturbances, pain, irritable bowel syndrome,fibromyalgia, and changes in voiding are associatedwith disturbances in brain circuits using specific neuro-transmitters, in particular serotonin or 5-hydroxytrypta-mine (5-HT). Fibromyalgia and irritable bowel syndro-me are conditions seen more often in patients withinterstitial cystitis (IC) than the general population

[510-514], and this condition is associated with an ove-ractive bladder and possibly to depression, which pro-vides a potential link with 5-HT metabolism. Perhapsthe strongest evidence for diminished 5-HT function indepressed patients is the remarkable efficacy of selecti-ve serotonin uptake inhibitors (SSRIs) in this group ofpatients. In addition, neuropharmacologic evidenceindicates that some forms of depression are associatedwith abnormalities in the promoter for the serotonintransporter gene [515,516].

5-HT has also been implicated in the regulation of painand also has a profound effect on bladder function.Neurons originating in the brainstem raphe nucleussynapse on visceral afferents and preganglionics in thethoracolumbar and sacral spinal cords. These neuronsrelease 5-HT. Stimulation of raphe nuclei in the brainstem inhibits reflex bladder contractions [517]. 5-HTand its precursors, uptake blockers or 5-HT analogsinhibit bladder activity in a variety of species [518].The actions of 5-HT are complex and mediated by over13 different receptors. Some data suggest that 5-HT hasa facilitating effect on voiding via modulation of blad-der afferents, but intrathecal antagonists of 5-HT2 and5-HT3 receptors reduce the volume threshold for voi-ding [519]. This latter data suggests that descending 5-HT pathways tonically depress bladder afferent input tothe sacral spinal cord. Moreover, the 5HT2C agonistsMCPP and MK212 inhibit isovolumetric rhythmic

142

Table 10 : Eight Na channel isoforms based on expression of a subunit. 6 isoforms at tetrodotoxin sensitive (TTX-S) and twoare resistant (TTX-R). With conditions such as peripheral nerve axotomy, inflammation, cystitis and in the SHR changes inNa currents are measured using patch clamp methods which serve as a model for peripheral terminal function. Some TTX-Risoforms are responsive to NGF, other GDNF. In transgenic mice over-expressing NGF or in DRG cultured in the presence ofNGF a rise in TTX-S currents is seen.

bladder contractions [481,520]. On the other hand, 5-HT1A antagonists inhibit bladder activity, probably onthe basis of prejunctional effects [521]. Outside theCNS, 5-HT influences neurotransmission to the blad-der. Presynaptic 5-HT receptors facilitate electricallyevoked bladder contractions in multiple species. Inhumans this facilitation is mediated by 5-HT4 receptors[522]. Taken together, the pharmacologic data suggestthat 5-HT in the CNS inhibits micturition reflex path-ways. By analogy, a genomic proclivity toward reduced5-HT neurotransmission in the CNS may lead to enhan-ced bladder activity.

The association between emotional disorders and blad-der complaints has been reported. Zorn and colleagues[523] studied 156 consecutive patients presenting to anincontinence clinic and compared their rates of depres-sion to a cohort of continent patients. A self-administe-red BeckÕs Depression Inventory (BDI) was used todiagnose depression and subjects were queried for ahistory of treatment for this condition. A third of incon-tinent patients had an abnormal BDI (>12) and/or a his-tory of depression, compared to only 17% of the conti-nent cohort group. Several patients were even referredto psychiatry because of the risk of suicidal behavior.The type of incontinence was stratified by physical exa-mination and urodynamics. Only 13% of patients withstress incontinence had depression, equivalent to thecontrol cohort. In contrast, 42% of patients with mixedor urge incontinence had depression by history and/orBDI scoring. Most notably, 60% of patients with idio-pathic urge incontinence had either undergone treat-ment for depression prior to the development of incon-tinence or scored in the depressive range on the BDI.This report suggests that the association betweendepression and mixed or urge incontinence is signifi-cant, but not with stress urinary incontinence. Onemight argue that patients with incontinence are merelymore likely to be depressed or undergo psychiatric eva-luation. These scenarios are unlikely because untreatedpatients with stress incontinence were no more likely tobe depressed than controls. Moreover, a significantassociation between depression and urge incontinenceexisted regardless of whether history of treatment orBDI score was used to categorize subjects. Othercauses of urge incontinence were not likely to be asso-ciated with depression.

Does correction of incontinence relieve depression?Recent studies examining BDI scores or symptoms ofdepression in women before and after surgery for stressincontinence revealed elevated postoperative scoreseven when incontinence was corrected [524,525]. Spe-cifically, Rosenzweig and colleagues reported thatdepression progressed after unsuccessful surgery, butwas not statistically improved with a successful proce-dure [524]. Together these findings suggest that incon-

tinence itself is not the sole cause of the associateddepression.

In this regard the model of depression associated withreduced levels of 5-HT in the brain has been shown tohave lowered volume threshold for voiding and uns-table bladder contractions. To clarify the link betweenemotional states, 5-HT and bladder function, we exa-mined rats depleted of 5-HT by neonatal treatment withclomipramine. These animals develop depression andhyperactive bladder. This bladder overactivity persistsin females but not males following puberty, a provoca-tive result

6. GENDER

Recent reviews suggest that urge incontinence anddepression are more common in women than men, andthat both conditions are commoner at times of changinghormonal levels in women [526]. Genetic, psychosocialand biological factors seem to account for this sexualdichotomy. One biological difference may be the func-tioning of 5-HT neurons. The serotonergic system playsa major role in depression, although there are other neu-rotransmitters involved. Differences in 5-HT functionmay explain why depression is more common inwomen. Women may be predisposed to depression inpart because levels of 5-HT in the brain are substantial-ly lower in women than men [527]. When the rates of5-HT synthesis were measured in the human brainusing PET, the mean rate of synthesis in normal maleswas found to be 52% higher than in normal females.Interestingly, estradiol (E2) administered to ovariecto-mized rats increases the density of 5-HT2A sites in thefrontal cortex, anterior cingulated gyrus and lateral dor-sal raphe. The areas with lowered 5-HT function onPET scanning correspond to areas of altered perfusionon PET scans during instructions to hold urine [342].Taken together, these data circumstantially support arole for reduced 5-HT activity or receptor function, hor-monal influences and linkage between brain sites fordepression, hyperactive voiding and altered 5-HT acti-vity.

In the CNS, estrogen modulates neuronal function byseveral mechanisms. One mechanism involves genomicactivation through the estrogen receptor, ER-a. ER-aimmunoreactive neurons are present in distinct areas ofthe forebrain, brainstem, and spinal cord linked withmicturition. The sacral spinal cord is one of the areasinvolved in micturition and in primates it contains ER-a immunoreactive neurons. Also, 5-HT neurons expressnuclear-estrogen receptor (ER-b) and progesteronereceptors (PRs), which are gene-transcription factors.SERT mRNA decreases with estrogen treatment in pri-mates [528]. Progesterone is without effect. Taken toge-ther, these data indicate that ovarian hormones could

143

modify cognition, emotions, pain, and autonomic neu-ral functions by acting on 5-HT neural pathways. Thus,the elimination of estrogens following ovariectomymight diminish the effect of neonatal clomipraminetreatment.

Since the last meeting there has been an exponentialincrease in the availability of animals in which geneshave been deleted. These knockout animals provideinformation concerning the role of the gene products inthe normal physiology and potentially in the pathophy-siology of the lower urinary tract. Studies on the P2X3receptor have been discussed above, but other impor-tant knockout animals have been studied and Table 11indicates some of those that have been studied in nor-mal animals.

The main conclusions from studies of knockout micehave been that the P2X3 receptor in pelvic nerve affe-rents that respond to physiological levels of distensionis essential for normal function in the afferent limb ofthe micturition reflex [63,67,68]. In the autonomic gan-glia, interference with the a subunit of the neuronalnicotinic receptor causes a failure of transmissionthrough these ganglia and results in increased bladdercapacity. Not surprisingly, the M3 receptor knockoutsuffers from urinary retention (predominantly in maleanimals). Of some interest also is the role of Insulin-like growth factor binding proteins, overexpression ofone of which cause bladder hypoplasia, and a humansyndrome linked to this is also known, if rare. Insulin-like growth factors influence the development of smoo-th muscle and blood vessels, and this finding is of inter-est to studies on development and of angiogenesis[529]. This is one example of the way in which trans-genic technology and human genomics can interact.The susceptibility of humans to pathology, and theways in which they cope with disturbances such aslower urinary tract obstruction may have its basis intheir genotype. It appears that animals without neuronalnitric oxide synthase can have normal lower urinarytract function, but inducible NOS is expressed afterbladder outlet obstruction, and knockout animals thatdo not possess inducible NOS are less able to cope withlower urinary tract obstruction [76].

Another gene (NCX) appears to control the number ofneurones innervating the bladder, and its deletionactually increases the number of NADP-diaphorasestaining cells in the autonomic ganglia. Bladder capaci-ty seems unaffected by deletion of the genes for neuro-

nal NOS, Brain Derived Neurotrophic Factor andcGMP-dependent protein kinase I (cGKI). HowevercGKI is involved in intracellular signaling followingrelease of nitric oxide, and deletion of this gene abo-lishes NO-cGMP-dependent relaxations of urethralsmooth muscle and results in hyperactive voiding[530].

The sensitivity of bladder smooth muscle to cholinergicstimulation appears to be reduced slightly in M2 recep-tor knockouts. Tonic, but not phasic, bladder contrac-tions are affected by knocking out smooth muscleheavy chain myosin, whereas contractile activityappears to be unaffected by deletion of the actin-bin-ding protein calponin.

Bladder sensation is reduced in animals that have had adeletion of the P2X3 or NK-1 receptor genes. In the for-mer case the bladder size is increased, whereas there areno reports of this in the latter instance. Pain sensitivitygenerally is reduced in deletions of the P2X2, NK-1,H1 and VR-1 receptor genes, although there appears tobe no information on other effects on the lower urinarytract. The significance of the P2X3 knockout in itseffects on the lower urinary tract appears to relate to thesignificance of this receptor in normal bladder function,i.e. it is involved in the transduction of normal pres-sures and volumes in the mouse bladder.

So the recent data on the effects of genes on lower uri-nary tract function is a growth area that will affect allaspects of lower urinary tract physiology, pharmacolo-gy and pathophysiology, and considerable growth inthis area can be expected.

Without discounting the importance of alterations insmooth muscle excitability, it appears that changes inbladder innervation orchestrated by neurotrophinsmanufactured by detrusor smooth muscle are temporal-ly linked with bladder overactivity. Direct proof inhumans is difficult to obtain, but circumstantial eviden-ce is compelling . The ability of local anesthetics, intra-vesical afferent neurotoxins, and destruction of afferentnerves in the bladder neck and prostate to reduce urgen-cy, frequency, and urge incontinence indicates animportant role for afferent evoked reflexes [531]. Thedevelopment of a spinal reflex (positive ice-water test)in patients with neurogenic bladders [479] as well as inpatients with bladder outlet obstruction [598] suggestsa common underlying plasticity in nerves supplying thebladder. Moreover, the association between elevatedblood pressure and lower urinary tract symptoms in

IV. SUMMARY OF THE NEUROGENICMECHANISMS UNDERLYINGBLADDER OVERACTIVITY

III. GENES AND THE REGULATIONOF LOWER URINARY TRACT

FUNCTION

144

patients with BPH [532] provides a link betweenchanges in sympathetic tone and voiding complaints. Inaddition the observation that aging is associated with anenhanced noradrenergic tone links the SHR model toplasticity in the lower urinary tract . Thus, commonthemes emerge when examining pathologic models.Urinary frequency and possibly urge incontinence areoften associated with elevated neurotrophin production,an enhanced C-fiber afferent evoked bladder reflex andnoradrenergic function. The realization that nerves sup-plying the lower urinary tract can undergo long-termchanges, which possibly lead to bladder overactivity,offers a novel avenue for therapeutic intervention.

The lower urinary tract has two main functions : stora-ge and periodic elimination of urine. These functionsare regulated by a complex neural control system loca-ted in the brain and spinal cord. This control systemperforms like a simple switching circuit to maintain areciprocal relationship between the reservoir (urinarybladder) and the outlet components of the urinary tract

(urethra and urethral sphincter). The switching circuit ismodulated by various neurtransmitters and is sensitiveto a variety of drugs. In infants the switching circuitsfunction in a purely reflex manner to produce involun-tary voiding; however in adults urine storage and relea-se are subject to voluntary control.

Injuries or diseases of the nervous system in adults candisrupt the voluntary control of micturition causing thereemergence of reflex micturition, resulting in bladderhyperactivity and incontinence. Because of the com-plexity of the central nervous control of the lower uri-nary tract, incontinence can occur as a result of a varie-ty of neurological disorders. Experimental studies indi-cate that bladder hyperactivity occurs after: (1) inter-ruption of cortical inhibitory circuits, (2) disruption ofbasal ganglia function in models of ParkinsonÕs disea-se, (3) damage to pathways from the brain to the spinalcord (multiple sclerosis, spinal cord injury) and (4) sen-sitization of bladder afferents. Various mechanismscontribute to the emergence of bladder dysfunctionincluding reorganisation of synaptic connections in thespinal cord, changes in the expression of neurotrans-mitters and receptors and alterations in neural-targetorgan interactions mediated by neurotrophic factors.

E. SUMMARY

145

Table 11 : Effects of Various knockouts on Bladder Function. The Table indicates which gene knockouts can influenceBladder size, Sensation, bladder contractions and neuronal targets in the manner indicated.

Increased Unchanged Decreased

Bladder Size P2X3 nNOS Insulin-like growth factor-bindingM3 (urinary retention in males) cGMP-dependent protein 4 (IGFBP-4) overexpressionAlpha3 subunit of the neuronal protein kinase I (cGKI) Ncx/Hox11L.1nicotinic acetylcholine receptor Brain Derived

Neurotrophic Factor (BDNF)

CMG or Bladder P2X3 M4 (M2)contractions M3 Calponin Smooth muscle myosin heavy

chain (tonic contraction)Alpha3 subunit of the neuronalnicotinic acetylcholine receptor

Sensation Purinergic P2XVanilloid VR-1Neurokinin NK1Histamine H1

Nerve Interactions Ncx/Hox11L.1 (Increases P2X3 (lack of response to ATPnumbers of NADP and distension)Diaphorase-staining ganglion cells) Alpha3 subunit of the neuronal

nicotinic acetylcholine receptor(Decreased ganglionic transmission)

NK-1 receptor

(diminished axon reflexes; failure of chemo-attraction of mast cells)

Data from References 35,63,67,75,99,529,530, 577-584.

During the past few years research in the field of Neu-rourology has led to the development of new conceptsregarding the etiology of hyperactive bladder dysfunc-tion and has stimulated the development of new thera-peutic approaches for the treatment of incontinence,including the intravesical administration of afferentneurotoxins, such as capsaicin . In future research itwill be important to focus on the development of neu-ropharmacologic agents that can suppress the uniquecomponents of abnormal bladder reflex mechanismsand thereby act selectively to diminish symptomswithout altering normal voiding function.

First and foremost, more human data is required to eva-luate the relative importance of mechanisms that havebeen demonstrated in animals. To an increasing extent,animal data is derived from studies of living cells, andthat should make it easier to make the required compa-risons. Nevertheless, whole animal and whole humanstudies will be essential if we are to extend our know-ledge of pathophysiology, where the interactions bet-ween tissue components often amplify the changes seenin cells. With all animal models we should never losesight of the question as to how closely these modelsreally mimic the human situation.

Studies of the rat major pelvic ganglion have been andwill continue to be useful in sorting out certain specificproblems in neurobiology, which might be expected tolead to further relevant developments, particularly inrelation to neuroplasticity. Nevertheless, the informa-tion that has been derived from such studies needs to beseen in the light of the human situation, where the gan-glia are distributed within the bladder wall, and easilysubjected to ischaemia and mechanical insults duringobstruction, overactive bladder and other conditionsthat might lead to partial denervation. Denervationaffects all intramural neurons and the effects of thesepathophysiological changes on afferents, pre- and post-ganglionic autonomic neurons and on intramural neu-rons and their interconnections is an area open forstudy. The growth factors that affect nerves and vessels(including the neurotrophins and insulin-related growthfactors and other trophic agents in angiogenesis and themaintenance of synaptic connectivity) each have speci-fic targets, and it is unlikely that nerve growth factor(neurotrophin-1) will be the only important molecule inthis area.

The neurotrophins however should remain a focus ofactivity partly because they are released by denervated

tissues, but also because they show such profoundchanges in inflammation, spinal cord injury and otherstates. They are at the center of the discussion concer-ning the relative roles of ischaemia and partial denerva-tion and of inflammation, cord injury and other patho-logies. There are many other potential mediators ashave been described earlier. In the related field of Spi-nal Cord Injury, there has recently been success in theuse of certain neurotrophins and of stem cells in facili-tating regeneration of central neurons, in some caseswith some return of function. There is increasing awa-reness that the nature of the trauma to the spinal cordcan alter the degree of damage, and new more appro-priate models are being used. There is now increasingactivity in this area and there can be added value in col-laboration with these workers.

Genetic susceptibility to disease is being understood toa greater extent as a result of the analysis of the humangenome. In this field comparisons with transgenic ani-mals will become increasingly important in basicresearch and genomics will provide information aboutthe susceptibility of humans to diseases. Transgenicanimals have recently been important in the discussionof factors which cause afferent hypersensitivity, and itwould be entirely appropriate to extend studies in theseareas to the expression of genes in patients with relateddiseases. For instance, do patients with interstitial cys-titis have a genetic susceptibility that might affect theirurothelium, their afferent sensitivity, their smoothmuscle, susceptibility of the tissues to iscahemia or avariety of other mechanisms that have been discussedin this volume. Genetic markers will be increasinglyimportant in humans, and it is already clear from ani-mal studies that tolerance of lower urinary tract obs-truction is aided by the presence of certain genes. Bythe next consultation, we hope that much more will beknown from animal and human studies to elucidatemechanisms and identify patients at particular risk.

These same genetic studies provide a new approach toneuropharmacology, and more specific therapies andappropriate preventive measures may become avai-lable. Studies of a more traditional nature continue tohave a major role in developments in pharmacology.

In short the continuing support for basic neuroscienceand cell biology, associated with the identification ofgenes and other factors that determine susceptibility todisease will provide a powerful approach that has, ulti-mately, to improve patient care. We are on the thresholdof a new era of relief for patients if sufficient funding isavailable and if we recruit the brightest individuals toimport the latest technologies to this important area.

F. FUTURE RESEARCH

146

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