Correlation between cystometric volumes, ATP release, and pH in women with overactive bladder versus controls.

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).

Comparison of receptor binding characteristics of commonly used muscarinic antagonists in human bladder detrusor and mucosa.

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.

Age-related changes of P2X(1) receptor mRNA in the bladder detrusor from men with and without bladder outlet obstruction.

The urinary bladder purinergic system is reported to change with age and with bladder dysfunction. Here, we examined the expression of purinergic P2X(1) receptors in detrusor and mucosa (urothelium+lamina propria) from male control bladder and investigated age-related P2X(1) receptor mRNA expression in control and obstructed detrusor. Biopsy specimens were obtained at cystoscopy from control patients (n=46, age range 30-86years) and patients diagnosed with outlet obstruction (n=29, 46-88years). Calponin expression (measured by RT-PCR) was similar in control and obstructed detrusor and did not change with age. Quantitative competitive RT-PCR was used to measure P2X(1) receptor and GAPDH mRNA in control and obstructed detrusor. P2X(1) receptor mRNA expression was 9-fold (p<0.0001) higher in the detrusor than in the mucosa. Expression of mRNA for the internal control GAPDH remained stable with age and across control and obstructed detrusor. No difference in P2X(1) receptor expression was observed between control and obstructed detrusor (p=0.35). However, an age-related decrease in P2X(1) mRNA expression was observed in control (n=27; p=0.0054; Spearman coefficient r=-0.520) but not obstructed detrusor (n=19; p=0.093; r=-0.396). Downregulation of P2X(1) mRNA expression might occur as a result of an increased component of neural ATP release in the aging bladder.

Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing.

1. We investigated muscarinic receptors in the detrusor and mucosa of the human bladder body. Radioligand-binding studies with [(3)H]QNB were conducted using specimens collected from patients (36-77 years) with normal bladder function, undergoing surgery. For RT-PCR, biopsies of normal bladder were obtained from patients (30-88 years) undergoing check cystoscopy. 2. Binding of [(3)H]QNB in detrusor (n=20) was of high affinity (K(D) 77.1 (55.2-99.0) pM) and capacity (B(max) 181+/-7 fmol mg protein(-1)). Similar values were obtained in mucosa (n=6) (K(D) 100.5 (41.2-159.9) pM; B(max) 145+/-9 fmol mg protein(-1)). 3. Competition-binding experiments in detrusor membranes with muscarinic receptor antagonists including trospium, darifenacin, 4-DAMP, methoctramine, AQ-RA 741, AF-DX 116 and pirenzepine indicated a receptor population of 71% M(2), 22% M(3) and 7% M(1). In the mucosa, 75% of sites were M(2) receptors, with 25% M(3)/M(5). 4. Using RT-PCR, expression of M(1), M(2), M(3) and M(5) mRNA was demonstrated in both detrusor and mucosa. 5. The presence of a high density of mainly M(2) muscarinic receptors in the mucosa appears to be a novel finding and raises the question of their physiological significance and the source of their endogenous ligand. 6. There was a negative correlation of receptor number (B(max)) with age in detrusor muscle from male patients (P=0.02). Quantitative competitive RT-PCR demonstrated a selective age-related decrease in mRNA for muscarinic M(3) but not M(2) receptors, in both male (P<0.0001) and female (P=0.019) detrusor. These findings correspond with reports of decreased detrusor contractility with ageing.

Enhancement of neurokinin A-induced smooth muscle contraction in human urinary bladder by mucosal removal and phosphoramidon: relationship to peptidase inhibition.

Neurokinin A (NKA) is potent in contracting the human detrusor muscle. Here, we have investigated whether these contractile responses are influenced by the presence of the mucosa, by the peptidase inhibitor phosphoramidon or by possible modulators, prostaglandins and nitric oxide. Contractile responses to neurokinin A were unaffected by indomethacin or N-omega-nitro-L-arginine, but were significantly reduced in strips containing mucosa. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11 (neprilysin, CD10), was ineffective at 10 microM, but at 100 microM, significant increase in the maximum response was achieved by neurokinin A in detrusor strips with and without mucosa. In immunohistochemical studies, neutral endopeptidase immunoreactivity occurred in peripheral nerve trunks in the detrusor and in a fibrous meshwork in the subepithelial lamina propria. Our data indicate that neutral endopeptidase is present in bladder mucosa and detrusor, and support the concept that this metalloprotease and/or related enzymes are important in regulating the actions of tachykinins.

Long-term darifenacin treatment for overactive bladder in patients aged 65 years and older: analysis of results from a 2-year, open-label extension study.

OBJECTIVES: This analysis evaluated the long-term safety, tolerability and efficacy of darifenacin, a muscarinic M3 selective receptor antagonist, in the treatment of overactive bladder (OAB) in patients > or = 65 years of age. METHODS: Patients who completed one of two 12-week, placebo-controlled, double-blind, feeder studies received once-daily (o.d.) treatment with darifenacin 7.5 mg for the first 2 weeks of the 2-year, open-label extension study. The dose could be subsequently adjusted (7.5 or 15 mg o.d.) according to need. Safety and tolerability were assessed, and efficacy variables/endpoints were evaluated from patient diary data. RESULTS: 214 patients (65-89 years) entered and 137 (64.0%) completed the 2-year extension study, amounting to 308 patient-years' drug exposure. Darifenacin was well tolerated with no new safety concerns. The most common adverse events (AEs) were dry mouth and constipation, which infrequently resulted in discontinuation (2.3% and 4.2%, respectively). Darifenacin produced significant improvements in OAB symptoms that were maintained over the 2-year period (median reduction from feeder-study baseline to 2 years: -11.0 [-83.7%] for incontinence episodes/week and -1.2 [-12.4%] for micturitions/day, both p < 0.05), with 44.4% patients achieving > or = 90% reduction in incontinence episodes at 2 years. CONCLUSIONS: Darifenacin demonstrated good tolerability and safety in older patients with OAB. The improvement in OAB symptoms was sustained throughout the 2-year extension, resulting in high treatment persistence rates. Results were comparable with those in the overall OAB population from this study, indicating that darifenacin treatment is effective and well tolerated irrespective of age.

Molecular characterization of M2 and M3 muscarinic receptor expression in bladder from women with refractory idiopathic detrusor overactivity.

OBJECTIVE: To examine the expression of muscarinic M2 and M3 receptors in human bladder detrusor and mucosa, from controls and patients with idiopathic detrusor overactivity (IDO), as antimuscarinic agents are the primary pharmacological treatment for IDO. PATIENTS AND METHODS: Biopsies from the bladder body were collected at cystoscopy from 20 women with urodynamically confirmed refractory IDO (age range 25-86 years); biopsies were also collected from 30 asymptomatic female controls (age range 32-87 years). Samples were collected into RNA extraction medium and dissected into mucosa (urothelium plus lamina propria) and detrusor. RNA was extracted and the expression of M2 and M3 receptor mRNA determined by quantitative competitive reverse transcription-polymerase chain reaction. Results were normalized to beta-actin expression in the same sample. RESULTS: Expression of M3 receptor mRNA, in mucosa of IDO patients (median 0.057 pg M3/100 ng total RNA; interquartile range 0.03-0.13, 12 samples), was four times (P = 0.039, Mann-Whitney) lower than from the control (median 0.22 pg M3/100 ng total RNA; 0.13-0.51, 11 samples). The expression of muscarinic M3 receptor mRNA was higher (14-35 times) in detrusor (control median 3.17; 26 samples) than in mucosa and did not change in IDO (median 2.03; 14 samples). M2 expression was not significantly different with region or with IDO. CONCLUSIONS: These data show that M3 muscarinic receptor mRNA expression was significantly less in mucosa from IDO patients than from age-matched controls. The role of mucosal M3 receptors is unknown at present and elucidation of this role might provide a greater understanding of the aetiology of IDO.

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

AIM: Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). MATERIALS AND METHODS: Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). RESULTS: TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. CONCLUSIONS: The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.