James I. Gillespie

Spontaneous microcontractions and electrical field stimulation (EFS)-evoked contractions in isolated rat bladder strips from normal and from 6 weeks partial bladder outflow obstruction (pBOO) animals were studied to identify the potential site of action for the β₃-adrenoceptor (AR) agonist mirabegron in detrusor overactivity in rats. For this, effects of the β-AR agonist isoprenaline and mirabegron were tested in presence or absence of selective antagonists for β-AR subtypes, namely CGP-20712A for β₁-AR, ICI-118,551 for β₂-AR, and L-748,337 for β₃-AR. In detrusor strips from both normal and obstructed animals, EFS-induced contractions were weakly affected by isoprenaline and even less so by mirabegron. In contrast, microcontraction activity was more potently reduced by isoprenaline (pIC50 7.3; Emax ±85 %), whereas mirabegron showed a small effect. In pBOO strips, concentration response curves for isoprenaline and mirabegron at inhibition of EFS and spontaneous microcontractions were similar to those in normal strips. Isoprenaline-induced inhibition of microcontractions and EFS was antagonized by the β₁-AR antagonist, but not by the β₂- and β₃-AR antagonists. In the context of β₃-AR-mediated bladder functions for mirabegron in other experiments, the current data question a role for effects at spontaneous microcontractions, or neurogenic detrusor stimulation in the mode of action for mirabegron in vivo, since functional bladder effects for mirabegron are reported to occur at much lower concentrations.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2 > M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small ‘micro’-contractions superposed on a tonic contraction, with immediate transient ‘rebound’ contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3 > M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology and pathology are discussed.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed.

Bladder afferent outflow, linked to sensation, plays a critical role in bladder pathology: abnormal outflow results in altered sensation, leading to increased voiding frequency, urge and often incontinence. β₃-adrenoceptor agonists have been suggested to be beneficial in treating these symptoms. However, the absence of a significant sympathetic innervation of the detrusor and only a modest relaxation of bladder muscle by β₃ agonists has questioned the therapeutic site of action of β₃ agonists in the bladder. The present study was done to explore the possibility that β₃-adrenoceptors might be located in the pelvic plexus. Using the rat, where the pelvic plexus is located primarily within a single ganglion, the major pelvic ganglion (MPG), immuno-histochemical approaches were used to identify structures expressing β₃-adrenoceptor immuno-reactivity (β₃AR-IR). The only structures found to express β₃AR-IR were small-diameter tyrosine hydroxylase and vesicular mono-amine transporter immuno-reactive (TH-IR and vmat-IR) neurones. These neurones, found in clusters or singly on the periphery of the ganglion, or dispersed in smaller clumps throughout the MPG, are similar to the small intensely fluorescent (SIF) cells described previously. Not all small cells expressed β₃AR-IR. A population of the small cells were also immuno-reactive to the type 3 muscarinic receptor (M₃R-IR) and the P2X3 purinergic receptor (P2X3-IR). Clumps of small cells were associated with calcitonin gene-related peptide immuno-reactive (CGRP-IR) nerve fibres (putative sensory fibres) and a small number were contacted by putative cholinergic nerves expressing immuno-reactivity to vesicular acetylcholine transporter (vacht-IR). These observations are consistent with the idea that small cells are interneurons and one of the components making up complex neural circuits within the MPG. The precise physiological role of these neural elements in the MPG is unknown. However, as one therapeutic action of β₃-adrenoceptor agonists is to modulate sensation, it is possible that these neural circuits may be involved in the regulation of afferent outflow and sensation.

What's known on the subject? and What does the study add? Experimental urethral obstruction in rats alters micturition patterns with non‐voiding activity (NVA) during filling cystometry, showing similarity to that observed in human detrusor overactivity. Several drug classes with therapeutic potential in overactive bladder in humans have been tested in this model in rats, rabbits or guinea pigs, but no detailed analysis of drug effects on cystometric patterns has been published. The present study uses a rat model of overactivity with partial bladder outflow obstruction (BOO) in combination with the procedures to analyse NVA to study the effects of the anticholinergic drug tolterodine and the novel β ₃ ‐adrenoceptor agonist mirabegron. The current data for the first time show that NVA in rats with BOO is sensitive to both the muscarinergic antagonist tolterodine and the β ₃ ‐adrenoceptor agonist mirabegron, but with clear differences between the two drugs: during progression of bladder filling, tolterodine affected both the amplitude and frequency of NVA whereas mirabegron affected primarily the frequency. In addition, tolterodine dose‐dependently reduced voiding contractions, while mirabegron did not. A model is proposed to account for these observations where both agents act on a ‘pacemaker‐like’ mechanism which is sensitive to cholinergic excitatory and beta‐adrenergic inhibitory inputs. Such concepts could provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs. OBJECTIVE: To investigate the hypothesis that tolterodine and the β ₃ ‐adrenoceptor agonist mirabegron exert their actions on the motor component of the motor/sensory system in the bladder wall: non‐voiding activity (NVA). MATERIALS AND METHODS: The present study used standard cystometric techniques and a conscious rat model of partial bladder outflow obstruction (BOO). A single dose of either tolterodine (0.01, 0.1 0.3 or 1.0 mg/kg) or mirabegron (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg) was given i.v. to each animal. RESULTS: In the dose ranges used, tolterodine reduced the voiding contraction amplitude, whereas mirabegron did not. Non‐voiding activity consisted of small (0.6 mmHg) transients. As a fill progressed, both tolterodine and mirabegron reduced the cumulative activity of the large non‐voiding contractions, but had little effect on the small transients. Tolterodine affected both the amplitude and frequency of NVA, whereas mirabegron affected primarily the frequency. CONCLUSIONS: Non‐voiding activity is sensitive to muscarinergic antagonists and β ₃ ‐adrenoceptor agonists, but there are clear differences between the two drugs. A model is proposed to account for these observations where both agents act on a ‘pacemaker‐like’ mechanism with cholinergic excitatory and adrenergic inhibitory inputs. Such concepts may provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs.

Study Type – Aetiology (case series) Level of Evidence 4 What's known on the subject? and What does the study add? The factors taken into consideration when determining when and where to void are poorly understood. Studies on bladder sensations, obtained during cystometry of from voiding diaries, are proving difficult to transfer to everyday experiences. There is therefore a need to explore what does influence when and where to void. This study, using focus groups, highlights the fact that many voids are driven by behavioural factors not by sensations of desire or need to void. It is further noted that a key factor in the final decision to void is an awareness of bladder volume. The concepts of ‘cognitive voiding’ informed by ‘bladder awareness’ are introduced and, if correct, will influence the way studies are devised and interpreted to explore lower urinary tract dysfunction and pharmacotherapy. OBJECTIVE To investigate the inter‐relationship between conscious decision‐making processes and bladder sensation in determining the need, time and place to void SUBJECTS AND METHODS: The approach used interview focus groups and qualitative thematic analysis. In this preliminary study, 25 women were included (aged 21–90 years) meeting in groups of one to five on four occasions. RESULTS: The thematic analysis yielded six themes: temporal and cognitive maps, risk issues, habituation and opportunistic behaviour and awareness of the bladder. For most voids, the decision to void was not based on sensation but determined by multiple factors: personal knowledge of time of last void, anticipated time to next void, proximity of toilets, a risk assessment or habituated behaviour. As the bladder filled the subjects described an increasing awareness of their bladder. Such sensations were not immediately associated with desire to void. Rather, these sensations were described as influencing the cognitive processes of considering when and where to void. A sub‐group of subjects reported little awareness as their bladder filled, experiencing only sudden strong sensations that needed immediate action for fear of leakage. CONCLUSIONS: The decision to void is primarily a cognitive process influenced by multiple elements of which bladder awareness is only one. Mechanisms generating awareness may be intensified or lost reflecting possible different pathological states. The importance of these observations in relation to current views of normal and abnormal voiding is discussed.

OBJECTIVE: To use an isolated preparation of the guinea‐pig bladder lamina propria (LP) to investigate the effects of adenosine tri‐phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E ₂ (PGE ₂ ). MATERIALS AND METHODS: The bladders of female guinea‐pigs (200–400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 °C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine‐NONOate (DEANO), and the effect on PGE ₂ output into the supernatant determined using the Parameter ^TM PGE ₂ enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. RESULTS: PGE ₂ production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P ₂ X and P ₂ Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE ₂ production over the initial 10 min of an exposure. However, PGE ₂ output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. CONCLUSIONS: There is complex interaction between ATP and NO to modulate PGE ₂ release from the bladder LP in the un‐stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.

Localized phasic contractions in the bladder wall (autonomous activity) have been hypothesized to be an integral part of a motor/sensory system contributing to bladder sensation. The sites responsible for generating this activity, the mechanisms involved in its propagation and modulation remain unknown. This phasic motor activity is modulated by exogenous prostaglandins. Therefore, analysis of the sites of prostaglandin production and action within the bladder wall may shed light on the mechanisms of generation and modulation of this phasic activity. In this paper we report the localization of immuno‐reactivity indicative of the expression of cyclo‐oxygenase enzyme type I (COX I‐IR) within the bladder wall. Basically, three types of COX I‐IR cell were identified: epithelial cells in the basal and intermediate layers of the urothelium, complex vimentin‐positive and COX I‐IR cells in the lamina propria and vimentin‐negative COX I‐IR cells in the lamina propria and on the surface of the inner muscle bundles. These vimentin‐negative/COX I‐IR cells appear to be in close apposition to a continuous network of vimentin‐positive cells, which extends from the lamina propria into the inner muscle layers and subsequently into the outer muscle layers. However, the interstitial cells in this region might form a distinctly different sub‐type. First, the interstitial cells in this region differ from those in the inner layer by their responsiveness to NO with a rise in cGMP. Two subtypes have been identified: cells on the surface of the muscle bundles and within the muscle bundles. Second, COX I‐IR cells are not associated with the interstitial cells in the outer layers. The physiological significance for these apparent differences in the interstitial cell network is not clear. However, such differences are likely to reflect differences in the processes involved in their activation, modulation and control.

OBJECTIVE: To examine the expression of ubiquitin hydrolase (UH), an enzyme which is part of the ubiquitin‐proteasome system involved in the regulation of cell growth and differentiation, to gain an insight into the cell types and processes underlying the tissue remodelling that occur after bladder neck damage. MATERIALS AND METHODS: Three groups of male guinea pigs were used, comprising controls (not operated, four), sham (five) and obstructed (six). The bladder outlet was obstructed by implanting a silver ring around the urethra, which was left in situ for 2–4 weeks. Sham‐operated guinea pigs had the same operative procedure but no ring was implanted. The bladders were removed and incubated in Krebs’ solution at 36 °C, gassed with 95% O ₂ and 5% CO ₂ , Tissues were then fixed in 4% depolymerized paraformaldehyde and processed for immunohistochemistry. We used antibodies raised against UH, cyclooxygenase type I and vimentin. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: Staining with an antibody to UH showed the presence of both sensory and motor nerves in control, sham and obstructed bladders. In the control bladders this was the predominant staining pattern. In the sham and obstructed bladders UH staining revealed additional positive cell types; cells associated with the outermost layers of the urothelium (the umbrella cells), in the lamina propria (the lamina propria interstitial cells (lp‐ICs), on the surface of the muscle bundles (surface muscle, sm‐ICs) and on the serosal surface (muscle coat, mc‐ICs). All ICs stained with vimentin. The ICs within the muscle bundles (intramuscular, im‐ICs) did not stain with UH. The number and density of the UH‐positive cells was greater in the obstructed than in the sham bladders, suggesting a change in relation to the severity of damage to the bladder neck. CONCLUSION: The expression of UH implies the re‐targeting of proteins marked for degradation in the proteasome. Increased expression of UH in the lp‐ICs and sm‐ICs shows that these cells are active in the early and late stages of the tissue remodelling resulting from obstruction. These results show a further subset of ICs that might be involved in the increased deposition of extracellular material and tissue remodelling.