Volume-induced effects on the isolated bladder - Wiley Online Library

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Helmut Klocker Jack Schalken Bill Watson Georg Bartsch David Neal Karl-Eric Andersson Kazem Azadzoi Olivier Cussenot Christopher Foster Robert Getzenberg Martin Gleave Hans Lilja Marston Linehan Norman Maitland Bruce Malkowicz Joel Nelson John Stein Ulf-Håkan Stenman Christian Stief George N. Thalmann Dan Theodorescu Tapio Visakorpi

BJU

INTERNATIONAL

EDITOR-IN-CHIEF JOHN M. FITZPATRICK

Blackwell Science, LtdOxford, UKBJUBJU International1464-410XBJU InternationalNovember 2004 947 Original Article LOCAL REFLEXES IN THE BLADDER WALL LAGOU et al.

Volume-induced effects on the isolated bladder: a possible local reflex MAGDALINI LAGOU, MARCUS J. DRAKE and JAMES I. GILLESPIE The Urophysiology Research Group, School of Surgical and Reproductive Sciences, The Medical School, The University, Newcastle upon Tyne, UK Accepted for publication 3 July 2004

OBJECTIVES To: (i) determine the effects of changing intravesical volume on autonomous activity in the isolated whole bladder of the guinea pig; (ii) identify the mechanisms which might contribute to induced changes; and (iii) explore the idea that changes in bladder volume which affect phasic activity are part of a local reflex operating within the bladder wall. MATERIALS AND METHODS Bladders were isolated from female guinea pigs, cannulated via the urethra and maintained in vitro in Tyrode’s solution. The intravesical pressure (IVP) was monitored and drugs added to the bathing solution. RESULTS The isolated unstimulated bladder containing 500–600 mL of fluid generates small (1–2 cmH2O) phasic rises in IVP, i.e. autonomous activity. When the bladder volume was increased, autonomous activity increased. In the presence of muscarinic agonists (100 nmol/L arecaidine and

INTRODUCTION The bladder wall is capable of generating complex coordinated activity which consists of transient rises in intravesical pressure (IVP), propagating waves of contraction and local stretches. Such activity has been described as ‘autonomous’ [1,2]. These phenomena have been examined in greatest detail in the guinea pig, but similar activity has been reported in the cat, dog, macaque, pig and rat [3–5]. In the guinea pig, autonomous activity is augmented by muscarinic and nicotinic agonists to produce larger rises in IVP, and more dynamic waves and stretches [2]. This 1356

carbachol 100 nmol/L) autonomous activity is augmented, giving rise to large (>10 cmH2O) phasic rises in IVP. When the volume was increased, both the amplitude and frequency of the transients increased. When the bladder volume was reduced there was a period of marked inhibition of phasic activity. To explore the mechanisms underlying these changes the possible involvement of local neural reflexes was explored. The neurotoxin tetrodotoxin had no effect on the volumeinduced changes. Sensory nerves are insensitive to tetrodotoxin and thus to assess their possible contribution bladders were exposed to capsaicin (10 mmol/L) to stimulate and eliminate sensory fibres; capsaicin caused complex changes in phasic activity, i.e. an initial increase, a secondary slowing and decrease, followed by a period of recovering amplitude and increased frequency. These changes suggest actions of the sensory nerves on the phasic mechanism indicative of a local axonal reflex. Once the phasic activity had returned to levels before capsaicin, changes in bladder volume still produced increases in activity and inhibition after the volume decrease. Interstitial cells (cells capable of increasing cGMP) are found in the bladder wall; to assess their possible role in the volume-induced changes, bladders were

treated with 30 mmol/L ODQ, an inhibitor of guanyl cyclase, for 30–60 min. The volumeinduced rise in frequency was little affected but the inhibition seen on volume reduction was reduced.

form of activity is similar to non-micturition phasic activity seen during the filling phase in many species, including man [3,6–10]. The augmented activity in the guinea pig can be modified by several different approaches. The frequency of the transients is increased by exposing the bladder to ATP analogues (a,bmethylene ATP), substance P and the nitric oxide donor sodium nitroprusside [11,12]. The frequency is also increased by electrical field stimulation (EFS) of the bladder nerves [2]. In contrast, the amplitude and frequency of the transients are decreased by noradrenaline [13]. These data may be interpreted to suggest that the mechanisms generating phasic

activity receive excitatory and inhibitory inputs [12,13].

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CONCLUSIONS These results show that there are components in the bladder wall which respond to distension by affecting phasic activity. This stimulus/response may reflect a volume ‘reflex’ within the bladder wall, consisting of excitatory and inhibitory components. This local reflex does not appear to involve directly motor or sensory nerves, although the latter can affect phasic activity, and their actions may represent a further reflex mechanism in the bladder wall. The possible involvement of guanyl cyclase in the volume-induced inhibition may indicate a role for interstitial cells. The physiological role of these mechanisms as a component of a motor/ sensor system in the bladder wall is discussed. KEYWORDS guinea pig, volume response, phasic activity, non micturition activity, local reflex, interstitial cells

The physiological role of the phasic activity and non-micturition contractions are unknown. Experiments on isolated pig bladder revealed small microcontractions, termed ‘micromotions’ [4]. Although not detected, it was suggested that these micromotions would give rise to microstretches, leading to activation of afferent nerves and sensation [4,14]. In the guinea pig the autonomous activity and the augmented activity are accompanied by local stretches of the bladder wall [1,2]. This directly supports the

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LOCAL REFLEXES IN THE BLADDER WALL

FIG. 1. The effects of progressive increments in IVV on autonomous activity in the isolated whole bladder of the guinea pig. A shows the original data. Where indicated by the stepped line the IVV was increased in 100 mL increments from an initial volume of 600 mL to 1300 mL. The volume was then returned to 600 mL. B shows on an expanded scale sections of the original record in A. (Data typical of a total of five experiments on five different bladders, showing the same result). 20 s A

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suggestion that phasic activity plays a role in generating sensation [10]. As the amplitude and frequency of the phasic activity can be increased by differing inputs, this suggests that bladder sensations can be modulated. It is therefore possible that there is a modulated motor/sensory system operating in the bladder wall [10]. One function of the bladder is to store urine. As the bladder volume increases this is signalled to the CNS so that appropriate action can be initiated. The accepted idea is that stretching the wall stimulates afferent nerves. In the modulated sensory system suggested above, sensations are also driven by phasic activity. It seems reasonable that if this system is involved in assessing collected urine, then bladder volume should also influence phasic activity and so affect sensation. In the present experiments we explored the effects of changing bladder volume on autonomous and augmented activity.

MATERIALS AND METHODS The procedures for isolating the whole bladder and maintaining it in vitro were

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described previously [1,2]. Briefly, female guinea pigs (12, 270–300 g) were killed by cervical dislocation in accordance with schedule 1 of the UK Home Office. The urinary bladder and urethra were removed and placed in Tyrode’s solution: (mmol/L): NaCl, 120; KCl 4.5; CaCl2 2.5; MgCl2 1; NaHCO3 25; NaH2PO4 1; Na pyruvate 1, glucose 5, and bubbled with 5% CO2 and 95% O2 (pH 7.4). The urethra was cannulated with a flexible plastic cannula (2 mm diameter) secured at the bladder neck using a fine ligature. Residual urine was gently removed using Tyrode’s solution. The bladder was then transferred to a heated organ bath (50 mL, 33–36 ∞C) containing constantly gassed Tyrode’s solution. The cannula was connected with the urethra uppermost via a fluid-filled tube and threeway connector to a pressure transducer (DTX Plus, Becton Dickinson, Milton Keynes, UK) and a 1-mL syringe to enable the intravesical volume (IVV) to be varied. The transducer output was amplified, digitized at 10 Hz and recorded using a capture system. The pressure range of this apparatus was 0.02–80 cmH2O. The transducer was calibrated before each experiment. At the outset each bladder was filled with Tyrode’s solution to give a baseline volume of 600–800 mL. These volumes were typical of the amounts of urine recovered

from animals immediately after cervical dislocation. In some animals the bladders were ‘empty’, i.e. containing 2000 mL. These basic observations set limits to the normal volume changes seen in guinea pigs of this size. The time taken from killing the animal to beginning recording was typically 30 min. Pressure recording was started immediately but the bladder was left to equilibrate for at least 20 min. The drugs used were the muscarinic agonist arecaidine but-2-ynyl ester tosylate, carbachol, tetrodotoxin, ODQ, and capsaicin (Tocris, UK). Concentrated drug solutions were added directly to the bath to achieve the required final dilution. All drugs were added to the solution bathing the abluminal surface. For EFS of the bladder wall, a pair of platinum electrodes was positioned on each side of the bladder in the organ bath. Applying short stimuli (1000 mL) for 100–200 s.

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to produce propagating waves of contraction [1]. In the presence of muscarinic agonists these transients were increased in both frequency and amplitude [2]. Therefore, the effects of volume were determined on this augmented activity.

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Thus further experiments were conducted to gain some insights into the possible components of this reflex in the bladder wall. One possibility is that volume is sensed by neuronal stretch receptors which relay

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information to the phasic mechanism and the detrusor via axons, and possibly the intramural ganglia. One way to explore this is to determine the actions of the neurotoxin tetrodotoxin; this blocks Na+ channels and so

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FIG. 4. The effect of different volume cycles on the phasic activity in an isolated whole guinea pig bladder. The bladder was exposed to a solution containing a mixture of 100 nmol/L each of arecaidine and carbachol. Where indicated the IVV was increased. Three step volume changes were given, i.e. 500 mL, 1000 mL and 1500 mL. The lower panel is an analysis of these data, showing changes in the instantaneous frequency plotted against time. The dotted horizontal line was drawn arbitrarily to illustrate the resting frequency before the volume increase. Note the undershoot in frequency each time the volume is decreased. +500 mL

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volume were used to assess any change in the responses. There was no systematic change, comparing responses before or after capsaicin (Fig. 7). If it is assumed that the afferent nerve function has been lost by this procedure, then this suggests that these nerves are not involved in generating the volume-induced responses, but they appear to have a modulatory role.


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The inhibition after a volume decrease is marked (Figs 4, 5, 7 and 8). One possible explanation for the loss of activity is that the bladder is flaccid and ‘floppy’ after a distension, and cannot generate force, or that any force generated does not result in an increase in IVP. To examine this possibility, bladders were subjected to high-frequency EFS (30 Hz, 0.5 ms pulses) to stimulate intrinsic nerves. At these frequencies cholinergic fibres are stimulated at the neuromuscular junction, resulting in a contraction of the whole bladder [2,19,20]. Bladders were then subjected to an increase and decrease in volume and the effects of EFS determined during these changes. Figure 8B shows one such experiment, typical of three others; the EFS responses after the volume decrease are not inhibited. In the experiment shown there may even be a small increase in the IVP generated. Thus, during the inhibition of the phasic activity the detrusor is responsive and the bladder is not excessively compliant.

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eliminates action potentials in neurones expressing these channels. Figure 5 shows that 1 mmol/L of tetrodotoxin had little effect on either the rise in frequency during a volume increase, or the inhibition resulting from a volume decrease. Thus, neurones and axons operating with tetrodotoxin-sensitive Na+ channels are not involved. It has been suggested that there are local axonic reflexes in many tissues which involve collaterals of afferent sensory fibres [15,16,17]. Sensory afferent nerves function using Na+ channels which are insensitive to tetrodotoxin [18]. Therefore, the operation of a mechanism involving sensory collaterals would not be affected by tetrodotoxin. To explore this possibility further, capsaicin was used. When it is applied to sensory nerves it has two actions, an initial stimulation,

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followed by nerve degeneration. When capsaicin was applied to isolated whole bladder preparations it resulted in complex changes in frequency and amplitude (Fig. 6). On initially applying capsaicin there was a transient rise in the frequency of the phasic activity. This was quickly followed by a period of inhibition, where the amplitude and frequency were decreased. With continued exposure the phasic activity recovered in amplitude, during which time there was a temporary rise in frequency. This response shows a possible excitatory and inhibitory influence of sensory afferents on phasic activity. Thus, there may be a local afferent axon collateral reflex functional in the bladder wall. Preparations were exposed to capsaicin for 30–120 min, during which step increases in

If tetrodotoxin-sensitive nerves and sensory afferents are not involved in the volumeinduced responses, then other mechanisms must be explored. There has been growing interest in the distribution and function of interstitial cells in the bladder wall. Two suggestions were proposed: (a) they are involved in some way in modulating sensation [21,22]; and (b) that they have a role in generating phasic activity [2,10,12]. Parts of the interstitial cell network in the bladder are capable of increasing cGMP in response to nitric oxide donors via stimulation of guanyl cyclase [12,23]. To explore the possible role of such cells in the volume-induced changes, bladders were treated with 30 mmol/L ODQ, an inhibitor of guanyl cyclase, for 30–60 min; Fig. 9 shows that ODQ had little effect on the rise in phasic activity during the volume increase, but reduced the inhibition seen after the volume decrease. Thus, cGMP-dependent mechanisms may be involved and this implies a role for interstitial cells. 1359

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FIG. 5. The effect of tetrodotoxin on the volume-induced responses in an isolated guinea pig bladder exposed to a solution containing 100 nmol/L each of arecaidine and carbachol. The upper panel shows original data from an experiment. The same result was obtained in four other preparations. Where indicated the IVV was increased by 1000 mL; 1 mmol/L tetrodotoxin was then added to the bathing solution and the volume change repeated. The lower panel shows an analysis of these data, showing the instantaneous frequency during the experiment.

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FIG. 6. The result of applying capsaicin (10 mmol/L) to the abluminal surface of an isolated whole bladder bathed in 100 nmol/L each of arecaidine and carbachol. The upper record shows original data from one experiment (typical of five others). The right inset shows a section of this record on an expanded time scale, illustrating the period of initial application of capsaicin. Where indicated, capsaicin was added to the bathing solution. The lower panel shows an analysis of these data, illustrating changes in instantaneous frequency plotted against time. The dotted horizontal line was drawn to illustrate the resting frequency before the volume increase.

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FIG. 8. The effects of one step change in IVV on an isolated whole bladder. A, Upper record shows original data from one experiment with the bladder bathed in a solution containing 100 nmol/L each of arecaidine and carbachol. Where indicated the IVV was increased from 600 mL to 1600 mL. The lower panel is an analysis of these data, showing changes in the instantaneous frequency plotted against time. The dotted horizontal line was drawn arbitrarily to illustrate the resting frequency before the volume increase. Note the undershoot in frequency when the bladder volume is decreased. B, shows data when the bladder was subjected to EFS (30 Hz, 5 s, 0.5 ms pulses) before, during and after a rise in IVV from 500 mL to 2000 mL; EFS was given where indicated by the inverted triangles. The volume was increased where shown by the horizontal bar, and the two periods of EFS during that time by the filled inverted triangles.

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FIG. 7. The absence of any effect of capsaicin pretreatment on the volume-induced changes in IVP in an isolated guinea pig bladder. A, upper panel (a), shows a control response in a bladder exposed to a mixed agonist solution containing 100 nmol/L each of arecaidine and carbachol. Where indicated the IVV was increases from 600 mL to 1900 mL in one step. The lower panel (b) shows an analysis of the response in (a) with the instantaneous frequency of the phasic activity. B(a) shows a section of record 60 min after exposure to 10 mmol/L capsaicin and (b) the analysis of this response. Data similar to these were obtained in five other preparations.

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Phasic activity is similar to non-micturition contractions seen in vivo during the filling phase of the micturition cycle in many species, including man. The physiological role of this form of activity is also unknown. It was postulated that local waves of contraction in the bladder wall could give rise to local stretches which activate mechanoreceptors in the wall, and so contribute to bladder sensations [4,10]. In this way the phenomenon of phasic non-micturition activity may form part of a motor/sensory system that can receive modulatory inputs from the CNS, i.e. a modulated sense organ [10]. The present data show that changes in the IVV increase and decrease the amplitude and frequency of the complex contractile activity. If the phasic activity is considered to be part of a motor/sensory system, it is an obvious extension of this concept to include an input derived from IVV. A key component is that the phasic mechanism is driven by a pacemaker, and it is here that inputs from different sources are integrated. In such a system a constantly varying sensory discharge would be generated. As the phasic mechanism also receives excitatory and inhibitory inputs 1362

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The present report describes a series of simple experiments which may have a profound effect on the way the organization and functions of the bladder wall are viewed. Recent work using isolated whole-bladder preparations of the guinea pig show that the bladder wall is capable of generating complex contractile activity in the absence of the CNS. The mechanisms generating these phasic contractions and their physiological significance are unknown [10]. Some have argued that they are not generated by the postganglionic parasympathetic innervation to the bladder, but rather the complexity of the responses, involving waves of contraction and local stretches of the wall, and insensitivity to atropine, has led to speculation that they may be generated within a heterogeneous network of cells, possibly involving interstitial cells and ganglia, with pacemaker regions and conductive pathways [2,10–14]. Data from the pharmacological interrogation of phasic activity have led to further speculation that the pacemaker mechanisms initiating the phasic activity also receive inputs from excitatory and inhibitory neural inputs [10].

FIG. 9. The actions of the guanyl cyclase inhibitor ODQ on the volume-induced changes in an isolated guinea pig bladder. A, shows a control response in a bladder bathed in a solution containing 100 nmol/L each of arecaidine and carbachol. Where indicated the IVV was increased. B, shows a response in the same bladder as A, 30 min after the preparation was exposed to 30 mmol/L ODQ. The changes in the recovery phase caused by ODQ after the volume increase were apparent in four preparations.

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[11,12], sensations related to volume may be influenced positively and negatively by these other inputs, e.g. by the CNS or local factors released from the urothelium. Thus, the sensation of IVV may be increased or decreased by mechanisms operating in the periphery. The inhibition of activity seen after a decrease in IVV was unexpected and its role is unknown. Once the bladder has been emptied after micturition it may be advantageous to eliminate or reduce sensory discharge. Such peripheral information

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processing may be important in the integration of signals by the CNS. Overall, afferent discharge from the bladder may be generated and modulated by several interrelated mechanisms, as shown in Fig. 10; (A) a ‘classical’ route whereby receptors in the wall activate afferent fibres in proportion to stretch in the bladder wall; (B) modulation of afferent discharge by agents released by the urothelium; and (C) local stretches modulated by phasic motor activity in the bladder wall.

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FIG. 10. A schematic diagram of the mechanisms which might contribute to the generation of sensation in the bladder. A, represents the ‘classical’ view of sensory nerve ending, deformed by stretch in the bladder wall, sending signals to the CNS. B, represents an extension to this concept, whereby substances released by the urothelium (nitric oxide, ATP inter alia) act directly on the afferent nerve ending to modulate discharge. C, represents the system outlined in the present report, proposing the existence of specific mechanisms within the bladder wall capable of generating phasic activity. This phasic activity or pacemaker controls a network of cells resulting in detrusor activation, which comprises contraction waves and local stretches. These stretches may activate afferent discharge [10]. The amplitude and frequency of the phasic activity can be influenced by different mechanisms, including the IVV, possibly via local reflexes, collaterals of sensory fibres and by excitatory and inhibitory inputs from the CNS. In this way such a complex mechanism could function as a modulated motor/sensory system.

present data are consistent with such a micro-anatomical arrangement in the bladder wall, whereby sensory collaterals innervate the mechanism determining the frequency of the phasic activity, the pacemaker mechanism. Possibly there is a sensory-axon collateral reflex in the bladder wall, the function of which is to modulate phasic activity. This concept is interesting in that it might be one indication of the physiological role of neurotransmitter release from sensory nerve endings in the bladder wall.

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Arguments in support of the final possibility have been rehearsed elsewhere [10]. Whether this overview of the complex sensory elements within the bladder wall is correct or not must await further experiments. The volume-induced changes in motor activity can be described as a simple reflex involving a sensor, an integration point and an output. The cellular components that comprise this reflex are unknown. The observation that tetrodotoxin and pretreatment with capsaicin have no effect on the volume responses may be an important clue in identifying these mechanisms. Tetrodotoxin will eliminate any contribution from nerves operating with tetrodotoxinsensitive Na+ channels. That tetrodotoxin has no action on the volume-induced changes implies that these nerves have little or no role in the basic mechanism. There are sensory nerve fibres which are insensitive to tetrodotoxin [18]; consequently, tetrodotoxin would not reveal a role for these sensory nerve fibres in the volume responses. It was

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for this reason that in the experiments with capsaicin we explored whether sensory nerves might contribute to the volume response. Capsaicin initially stimulates sensory nerve fibres to release their complement of neurotransmitters and, with prolonged exposure, leads to loss of sensation and ultimately axon elimination [15]. In the bladder the sensory nerves contain substance P and calcitonin gene-related peptide (CGRP) [15–17]. Interestingly, no integrated physiological mechanisms involving the release of these transmitters from sensory nerves in the bladder have been unequivocally reported. The acute effects of applied capsaicin on the phasic activity may arise from its action on the sensory fibres and the release of their transmitter content. In keeping with this possibility, substance P increases the frequency of phasic activity [11], while CGRP has a marked inhibitory action (Gillespie, in preparation). It has been known for over a century that afferent nerves can send collateral fibres to structures within the tissue where they originate [15–17]. The

The urothelium releases ATP in response to increased hydrostatic pressure [24]. It was suggested that the physiological role of this urothelial ATP is to modulate the firing of afferent nerve fibres, and in so doing to influence bladder sensation [24–26]. In the whole bladder preparation, exogenously applied ATP analogue (a,b-methylene ATP) has little effect on the resting bladder, but has a marked effect on the phasic activity [10]. Data such as this led to the suggestion that ATP has an indirect action in the bladder, acting via the phasic activity. The present data suggest a further possibility, that the actions of exogenous and endogenous ATP on motor activity are via sensory fibres. In this way, stretch of the urothelium to releases ATP would inducing firing in the afferent fibres. Antidromic activation of collateral fibres of these nerves could activate the phasic mechanism by releasing, e.g. substance P. Such a mechanism might contribute to the excitation of phasic activity seen during the increases in IVV. Prolonged exposure to capsaicin leads to the elimination of sensory nerves [15]; in the present experiments exposure to capsaicin for 30–60 min produced temporary effects on the phasic activity. Exposure to capsaicin for such short times is effective in activating sensory fibres, resulting in their functional removal [27]. Once the phasic activity had recovered from the acute exposure to capsaicin, the volume-induced changes were unaffected. This suggests that the local sensory collateral reflex is not involved in the volume response. If all neural sensory influences can be eliminated then other, nonneural, mechanisms must be involved. That ODQ, an inhibitor of guanyl cyclase, affects the inhibitory phase induced by a volume decrease suggests an involvement of cells signalling via cGMP. In the bladder wall there are several cell types which are capable 1363

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of producing increases in cGMP [23]. There is a small population (