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ATP is an important mediator of urgency in women with detrusor overactivity (DO). In order to understand how different degrees of bladder stretch elicited ATP release in DO patients compared with controls, sequential aliquots were collected during cystometry and ATP release was measured at each degree of bladder filling, in female patients with DO and controls. In both DO and control groups, ATP release was induced during bladder filling, suggesting that stretch stimulated further ATP release. However, the luminal ATP concentrations were already high at early filling stage (200 mL), which was even greater than those at the later filling stages (400 mL and maximum cystometric capacity, MCC), indicating that a substantial ATP release has been induced during early filling (200 mL) in both DO and controls. In DO, ATP release at 200 mL was significantly higher in those with low first desire to void (FDV) (≤200 mL) than in those with higher FDV (>200 mL); this may suggest that ATP release at early stretch may play an important role in urgency (early sensation) in DO. ATP concentrations remained unchanged after voiding, suggesting that voiding did not further induce ATP release into intraluminal fluid.

AIMS: In the bladder, ATP is an important signaling molecule, which is released by bladder stretch and acid. We hypothesized that ATP might play a unique role in patients with OAB, characterized by low bladder volumes at first desire to void (FDV) and maximal cystometric capacity (MCC) and symptoms of frequency/urgency [mild bladder pain syndrome (BPS)]. Our aim was to investigate the correlation between ATP release and urodynamic parameters, as well as urine pH, in OAB patients. METHODS: Routine cystometry was performed in a consecutive series of 249 women. The voided urodynamic fluid (VUF) was stored at -20°C and ATP measured using bioluminescence. Catheter urine was collected for pH measurement. Correlations between two factors were tested by linear regression analysis. RESULTS: Subjects with urinary tract infection, voiding dysfunction, and detrusor overactivity (DO) were excluded. For OAB patients (n = 25), there was an inverse correlation between ATP concentration in VUF and FDV (r(2) = 0.25; P = 0.01) but not MCC. This was not seen in controls (n = 69). In OAB, but not controls, there was a significant reverse correlation (r(2) = 0.16; P = 0.047) between ATP in VUF and urine pH. Urine pH was not significantly correlated with MCC in either group. CONCLUSIONS: In OAB patients, ATP is an important factor for initial perception of need to urinate (as indicated by FDV). This is similar to our previous findings in patients with DO, suggesting that ATP may mediate initial afferent sensation in patients with bladder dysfunctions characterized by urgency. ATP release was also strongly affected by urine pH, in patients with OAB (at FDV). Neurourol. Urodynam.

INTRODUCTION AND OBJECTIVES: Patients with bladder oversensitivity (80) (previously called painful bladder syndrome, PBS) suffer from urgency and frequency, often accompanied by suprapubic pain. Because ATP is an important sig nall ing molecule in bladder fun ction and dysfunction, ATP might playa unique role in women wi th 80. Our aim was to investigate the correlati on between ATP release and urodynamic parameters, Le., first desire to void (FDV) and maximum cystometry capacity (MCG) in BO patients, as welt as urine pH. METHODS: Routine cystometry was performed in a consecutive series of 237 women, 28 women were diagnosed wi th BO. 4 patients with urinary tract infection were excluded . The voided urodynamic fluid was stored at -200C and ATP measured using a bioluminescence assay. Midstream urine was collected for pH measurement. Statistical analysis was performed using GraphPad Pri sm 5.0 software. Correlations between two factors were tested by linear regression analysis. RESULTS: There was a significan t reverse correlation between the concentration of ATP in bladder washings and FDV (r2= 0.25; P= 0.013) (Fig 1). The correlation between ATP concentration and MCC (P= 0.32) or voided volume (W) (P = 0.31) was not significant. There was a significant reverse correlation between ATP concentration in voided urodynamic fluid and the urine pH (Fig 2). CONCLUSIONS: ATP is an important factor for initial perception of need to urinate (as indicated by FDV). This is similar to our previous findings in patients with detrusor overactivity, suggesting that ATP may mediate initial afferent sensation in patien ts with bladder dysfunctions characterised by urgency. ATP release of BO patients was also strongly affected by their urine pH.

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.

In vitro animal studies have shown that increasing acidity, within the physiological range, will enhance release of ATP from the bladder [1]. Here, we explored the hypothesis that in vivo urinary pH would affect ATP release during bladder filling. The aim of this study was to test whether ATP release in bladder during stretch is related to the patient’s urine pH.

It is now well accepted that ATP released from the bladder urothelium in response to stretch [1] activates underlying afferent nerves and signals bladder fullness. Furthermore it is well known that low pH can stimulate ATP release in vitro [2]. In patients with Detrusor overactivity (DO) an inverse correlation between the intravesical concentration of ATP and the bladder volume at first desire to void (FDV) has previously been reported [3]. A similar correlation was not reported in control patients [3]. These correlations were seen in voided urodynamic fluid taken at the end of the cystometry test. The possibility that ATP was released during the voiding bladder contraction could not be evaluated. The aim of our study was to 1) examine the concentration of ATP in intravesical fluid aliquots taken at bladder volumes of 200 and 400ml during filling cystometry and 2) to examine urinary pH in relation to ATP concentration in intravesical fluid.

Background: The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation. Methods: Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence. Results: Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients’ bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (aSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblastlike cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections. Conclusions: Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.

PurposeAdenosine triphosphate released from urothelium during stretch stimulates afferent nerves and conveys information on bladder fullness. We measured adenosine triphosphate released during cystometric bladder filling in women with idiopathic detrusor overactivity and stress incontinence (controls), and assessed whether the level of released adenosine triphosphate is related to cystometric parameters.Materials and MethodsRoutine cystometry was done in 51 controls and 48 women with detrusor overactivity who were 28 to 87 years old. Voided urodynamic fluid was collected and stored at ¿30C. Adenosine triphosphate was measured by a bioluminescence assay.ResultsAdenosine triphosphate levels were similar in voided urodynamic fluid of controls and patients with detrusor overactivity (p = 0.79). A significant inverse correlation was seen between adenosine triphosphate and maximal cystometric capacity in controls (p = 0.013), and between voided volume and adenosine triphosphate in controls (p = 0.015) and detrusor overactivity cases (p = 0.019). A significant correlation between first desire to void and adenosine triphosphate was also noted in detrusor overactivity cases (p = 0.033) but not in controls (p = 0.58). No correlation was seen between adenosine triphosphate and detrusor pressure during filling or voiding.ConclusionsAdenosine triphosphate measurement in voided urodynamic fluid is a novel approach to understanding signals that may contribute to the urgency sensation (a sudden compelling desire to pass urine). The inverse correlation between adenosine triphosphate in voided urodynamic fluid and first desire to void suggests that adenosine triphosphate has a role in modulating the early filling sensation in patients with detrusor overactivity.

ATP is an important signalling molecule whose release from the bladder urothelium in response to stretch is thought to stimulate afferent nerves and thus convey information about bladder fullness (and the sensation of urgency). Our aim was to measure the release of ATP during urodynamic filling of bladders of women with idiopathic detrusor overactivity (DO), painful bladder syndrome (PBS) and stress incontinence (controls), in order to test the hypothesis that ATP release might play a role in bladder function and/or dysfunction.

Hypothesis / aims of study: Urothelial ATP release is thought to play an important role in bladder afferent signaling via activation of purinergic receptors on suburothelial afferent nerves. Stretch of the bladder mucosa is a well documented stimulus for ATP release in several species, including the pig [1]. In addition, in mouse bladder, capsaicin is also an effective stimulus for ATP release, acting via stimulation of vanilloid (TRPV1) receptors [2]. While acid is an agonist at the TRPV1 receptor, specialized acid sensing ion channels (ASICs) are also present in several organ systems [3]. Our aim was to characterize the ATP release from pig bladder mucosa in response to stretch, acid and capsaicin.

Recent studies have described muscarinic receptors on the mucosa and the detrusor of the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear. Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine, and trospium. Specimens were collected from asymptomatic patients (50-72 years old) undergoing open bladder surgery. Radioligand binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were performed separately on detrusor and mucosal membranes. All antagonists displayed high affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition constants were also obtained for all antagonists against individual muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Here, fesoterodine showed anomalous binding results, suggesting that some conversion to its metabolite had occurred. Global nonlinear regression analysis of bladder binding data with five antagonists demonstrated 82% low-affinity sites in mucosa and 78% low-affinity sites in detrusor, probably representing M(2)/M(4) receptors. There was an excellent correlation (r(2) = 0.99) of low-affinity global estimates between detrusor and mucosa, whereas the corresponding high-affinity estimates ( approximately 20% of sites) were dissimilar. In conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to receptors located on the bladder mucosa and the detrusor, providing support for the hypothesis that muscarinic receptors in the mucosa may represent an important site of action for these agents in OAB.