Hypersensitivity of bladder low threshold, wide dynamic range, afferent fibres following treatment with the chemotherapeutic drugs cyclophosphamide and ifosfamide.

The cytotoxic drugs cyclophosphamide (CPO) and ifosfamide (IFO) cause toxic urological effects due to the production of urinary metabolites that cause bladder inflammation. This study aimed to identify changes in the bladder afferent system following treatment with these drugs that might explain reported urological adverse effects. Intravesical pressure and afferent nerve activity were recorded during bladder distension and drug administration in isolated bladders from mice, 24 h after intraperitoneal treatment with cyclophosphamide (100 mg/kg), ifosphamide (200 mg/kg) or saline (control). In isolated bladders, total afferent nerve activity at maximum bladder distension was increased from 182 ± 13 imp/s in control animals, to 230 ± 14 imp/s in CPO-treated (p < 0.05) and 226 ± 17 imp/s in IFO-treated (p < 0.001) mice. Single fibre analysis revealed the increase resulted from an enhanced activity in low threshold, wide dynamic range fibres (23.3 ± 1.9 imp/s/fibre in controls to 31.5 ± 2.5 (p < 0.01) in CPO and 29.9 ± 2.0 imp/s/fibre (p < 0.05) in IFO treated). CPO treatment was accompanied by an increase in urinary frequency in vivo, but was not associated with increases in urothelial release of ATP or acetylcholine, bladder compliance or spontaneous muscle activity. Also, CPO-treatment did not affect afferent nerve responses or pressure responses to purinergic, muscarinic or nicotinic agonists. This is the first report of CPO and IFO-induced changes in specific populations of bladder afferents, namely an increase in low threshold, wide dynamic range fibres. These effects appear to be direct and not secondary to increases in smooth muscle activity or the release of urothelial mediators.

α1L-adrenoceptors mediate contraction of human erectile tissue.

α1-adrenoceptor antagonists can impact upon sexual function and have potential in the treatment of erectile dysfunction. Human erectile tissue contains predominantly α1A-adrenoceptors, and here we examined whether contractions of this tissue are mediated by the functional phenotype, the α1L-adrenoceptor. Functional experiments using subtype selective agonists and antagonists, along with radioligand ([3H]tamsulosin) binding assays, were used to determine the α1-adrenoceptor population. A61603, a α1A-adrenoceptor agonist, was a full agonist with a potency 21-fold greater than that of noradrenaline. The α1A- and α1D-adrenoceptor antagonist tamsulosin antagonized noradrenaline responses with high affinity (pKD = 9.7 ± 0.3), whilst BMY7378 (100 nM) (α1D-adrenoceptor antagonist) failed to antagonize responses. In contrast, relatively low affinity estimates were obtained for both prazosin (pKD = 8.2 ± 0.1) and RS17053 (pKD = 6.9 ± 0.2), antagonists which discriminate between the α1A- and α1L-adrenoceptors. [3H]Tamsulosin bound with high affinity to the receptors of human erectile tissue (pKD = 10.3 ± 0.1) with a receptor density of 28.1 ± 1.4 fmol mg-1 protein. Prazosin displacement of [3H]tamsulosin binding revealed a single homogenous population of binding sites with a relatively low affinity for prazosin (pKi = 8.9). Taken together these data confirm that the receptor mediating contraction in human erectile tissue has the pharmacological properties of the α1L-adrenoceptor.

Diabetes-induced alterations in urothelium function: Enhanced ATP release and nerve-evoked contractions in the streptozotocin rat bladder.

Up to 80% of patients with diabetes mellitus develop lower urinary tract complications, most commonly diabetic bladder dysfunction (DBD). The aim of this study was to investigate the impact of diabetes on the function of the inner bladder lining (urothelium). Bladder compliance and intraluminal release of urothelial mediators, adenosine triphosphate (ATP) and acetylcholine (ACh) in response to distension were investigated in whole bladders isolated from 2- and 12-week streptozotocin (STZ)-diabetic rats. Intact and urothelium-denuded bladder strips were used to assess the influence of the urothelium on bladder contractility. Intraluminal ATP release was significantly enhanced at 2 weeks of diabetes, although not at 12 weeks. In contrast, intraluminal ACh release was unaltered by diabetes. Bladder compliance was also significantly enhanced at both 2 and 12 weeks of diabetes, with greatly reduced intravesical pressures in response to distension. Nerve-evoked contractions of bladder strips were significantly greater at 2 weeks of diabetes. When the urothelium was absent, nerve-evoked contractions were reduced, but contractions remained significantly elevated at lower frequencies of stimulation (<5 Hz) in diabetics. Interestingly, although relaxations of bladder strips to isoprenaline were unaltered by diabetes, removal of the urothelium unmasked significantly enhanced relaxations in strips from 2- and 12-week diabetic animals. In conclusion, diabetes alters urothelial function. Enhanced urothelial ATP release may be involved in the hypercontractility observed at early time points of diabetes. These alterations are time-dependent and may contribute to the mechanisms at play during the development of diabetic bladder dysfunction.

Inhibitory effect of the urothelium/lamina propria on female porcine urethral contractility & effect of age.

The urethral uroepithelium has been implicated in urethral sensation and maintenance of continence. However, relatively little is known about the function of the urethral urothelium compared with that of the bladder. The aim of the study was to examine the role of the urothelium/lamina propria on contractility of the porcine urethra, along with the influence of nitric oxide, prostaglandins and ageing. Porcine urethral tissues, intact and denuded of urothelium/lamina propria, were mounted in tissue baths and contractions to noradrenaline, phenylephrine and carbachol obtained. Contractions in the presence of Nώ-nitro-l-arginine (100 µmol/L) and indomethacin (10 µmol/L) were examined, along with contractions of tissues from young (6 months) and older (3 years) animals. The urothelium/lamina propria of the urethra significantly inhibited contractions to carbachol, noradrenaline and phenylephrine. This inhibitory effect was not significantly different for the three agonists (58.7±10.3%, 60.4±12.6% and 39.4±12.2% inhibition; n=4-7), and was also observed when denuded tissues were co-incubated with a second tissue with intact urothelium/lamina propria (40.6±7.5% inhibition; n=6). Inhibition of nitric oxide and prostaglandin production did not attenuate the inhibitory effect of the urothelium/lamina propria on noradrenaline contractions. In addition, ageing did not alter the inhibitory effect for either phenylephrine contractions (33.9±2.2% vs 41.0±9.7%, young vs older urethral tissues) or noradrenaline contractions (32.9±11.1% vs 53.7±11.0%). In conclusion the urothelium/lamina propria of the urethra has an inhibitory effect on receptor-mediated urethral contraction. This inhibition is due to the release of a diffusible factor, and the effect is not mediated by nitric oxide or prostaglandins, or affected by age.

Effect of Testosterone on Inflammatory Markers in the Development of Early Atherogenesis in the Testicular-Feminized Mouse Model.

BACKGROUND: Low levels of serum testosterone in men are associated with cardiovascular disease. Clinical studies show that testosterone replacement therapy (TRT) can improve symptoms of cardiovascular disease and reduce the inflammatory burden evident in atherosclerosis. AIM: We used an in vivo animal model to determine whether testosterone influences mediators of vascular inflammation as part of its beneficial effects on atherogenesis. METHODS: Testicular-feminized (Tfm) mice, which express low endogenous testosterone and a non-functional androgen receptor (AR), were used to assess the effect of androgen status on atheroma formation, serum lipids, and inflammatory mediators. Tfm mice were fed a high-cholesterol diet, received saline or physiological (TRT), and were compared to saline-treated XY littermates. RESULTS: A total of 28 weeks of high-cholesterol diet caused fatty streak formation in the aortic root of XY littermates and Tfm mice, an effect significantly amplified in Tfm mice. Tfm mice on normal diet showed elevated serum tumor necrosis factor-α (TFN-α) and interleukin-6 compared to XY littermates. High-cholesterol diet induced increased monocyte chemoattractant protein-1 (MCP-1) in Tfm mice, and TFN-α and MCP-1 in XY littermates. TRT reduced fatty streak formation and serum interleukin-6 in Tfm mice but had no significant effects on lipid profiles. Monocyte/macrophage staining indicated local inflammation in aortic root fatty streak areas of all mice, with TRT reducing local inflammation through plaque reduction in Tfm mice. Fractalkine (CX3CL1) and its receptor (CX3CR1) were present in fatty streaks of all mice fed a high-cholesterol diet, independent of androgen status. CONCLUSION: These results are consistent with AR-dependent and AR-independent anti-inflammatory actions of testosterone in atheroprotection, although the local anti-inflammatory mechanisms via which testosterone acts remain unknown.

Muscarinic agonists and antagonists: effects on the urinary bladder.

Voiding of the bladder is the result of a parasympathetic muscarinic receptor activation of the detrusor smooth muscle. However, the maintenance of continence and a normal bladder micturition cycle involves a complex interaction of cholinergic, adrenergic, nitrergic and peptidergic systems that is currently little understood. The cholinergic component of bladder control involves two systems, acetylcholine (ACh) released from parasympathetic nerves and ACh from non-neuronal cells within the urothelium. The actions of ACh on the bladder depend on the presence of muscarinic receptors that are located on the detrusor smooth muscle, where they cause direct (M3) and indirect (M2) contraction; pre-junctional nerve terminals where they increase (M1) or decrease (M4) the release of ACh and noradrenaline (NA); sensory nerves where they influence afferent nerve activity; umbrella cells in the urothelium where they stimulate the release of ATP and NO; suburothelial interstitial cells with unknown function; and finally, other unidentified sites in the urothelium from where prostaglandins and inhibitory/relaxatory factors are released. Thus, the actions of muscarinic receptor agonists and antagonists on the bladder may be very complex even when considering only local muscarinic actions. Clinically, muscarinic antagonists remain the mainstay of treatment for the overactive bladder (OAB), while muscarinic agonists have been used to treat hypoactive bladder. The antagonists are effective in treating OAB, but their precise mechanisms and sites of action (detrusor, urothelium, and nerves) have yet to be established. Potentially more selective agents may be developed when the cholinergic systems within the bladder are more fully understood.

Current and emerging pharmacological targets for medical expulsive therapy.

The primary goals of medical expulsive therapy are to increase the rate of stone expulsion along the ureter to avoid ureteral obstruction and reduce ureteral colic and thus avoid the need for surgical and more invasive interventions. This review focussed on the findings from in vivo and in vitro animal and human studies that have investigated the pharmacological mechanisms controlling ureteral motility and their translation to current and potentially new clinically used drugs for increasing the rate of stone expulsion along the ureter. The complicated contractility profile of the ureter, which alters with age, tissue segment region, orientation and species contributes to the difficulty of interpreting studies on ureteral pharmacology, which translates to the complexity of discovering ideal drug targets for medical expulsive therapy. Nevertheless, the current drug classes clinically used for patients with stone lodgement include α1 -adrenoceptor antagonists, calcium channel blockers and NSAIDS, whilst there are promising targets for drug development that require further clinical investigations including the phosphodiesterase type 5 enzyme, ß-adrenoceptors and 5-HT receptors.

Partial recovery of voiding function in female mice following repeated psychological stress exposure.

Psychological stress causes bladder dysfunction in humans and in rodent models, with increased urinary frequency and altered contractile responses evident following repeated environmental stress exposure. However, whether these changes persist after removal of the stressor is unknown, and the aim of this study was to determine if stress-induced changes in voiding behaviour and bladder function recover following removal of the stressor. Adult female mice were allocated to three groups: Unstressed, Stressed or Stressed + Recovery. Animals in the stressed groups were exposed to water avoidance stress for 1h/day for 10-days, with unstressed animals age-matched and housed under normal conditions. For recovery studies, animals were housed without stress exposure for an additional 10-days. Voiding behaviour was assessed periodically and animals sacrificed on day 10 (Unstressed and Stressed) or day 20 (Unstressed and Stressed + Recovery). Isolated whole bladder studies were used to assess compliance, urothelial mediator release and contractile responses. Exposure to stress increased plasma corticosterone levels almost three-fold (P<0.05) but this returned to baseline during the recovery period. Contractile responses of the bladder to carbachol and KCl were also increased following stress, and again fully recovered after a 10-day stress-free period. In contrast, stress increased urinary frequency four-fold (P<0.001), but this did not return fully to baseline during the recovery period. Bladder compliance was unchanged by stress; however, it was increased in the stressed + recovery group (P<0.05). Thus, following a stress-free period there is partial recovery of voiding behaviour, with an increase in bladder compliance possibly contributing to the compensatory mechanisms.

Mirabegron and solifenacin are effective for the management of the increased urinary frequency induced by psychological stress in female mice.

Evidence to support the effectiveness of ß3-adrenoceptor agonist mirabegron and anti-muscarinic solifenacin in the management of bladder dysfunction caused by psychological stress is lacking. This study investigates whether mirabegron or solifenacin reduces the bladder overactivity caused by water avoidance stress (WAS) in mice. Female mice were exposed to WAS for 1 h/day for 10 days and received either placebo, solifenacin or mirabegron in drinking water. Controls were age-matched without stress exposure. Voiding behaviour and functional isolated whole bladder responses during distension and in response to pharmacological agents and electrical field stimulation was investigated. Urinary frequency was significantly increased following stress. Mice treated with mirabegron or solifenacin displayed significantly fewer voiding events compared to the stressed mice, and voiding frequency in drug-treated animals was comparable to unstressed controls. The maximal contractile responses of bladders to carbachol were significantly enhanced by stress and reduced by mirabegron but not solifenacin. The frequency of phasic bladder contractions following stimulation with carbachol was significantly enhanced following stress and remained elevated in the mirabegron treated group. However, treatment with solifenacin significantly reduced the frequency of phasic contractions to unstressed control levels. Solifenacin and mirabegron are beneficial in reducing the overall voiding dysfunction caused by WAS in mice.

The role of c-kit-positive interstitial cells in mediating phasic contractions of bladder strips from streptozotocin-induced diabetic rats.

OBJECTIVE: • To investigate the role of c-kit-positive interstitial cells (ICCs) in mediating muscarinic receptor-induced phasic contractions of isolated bladder strips from streptozotocin(STZ)-induced diabetic rats and to confirm the expression and location of ICCs in the rat bladder. MATERIALS AND METHODS: • Bladders were removed from STZ-induced diabetic rats at 1, 4 and 12 weeks after induction of diabetes and from age-matched controls. • To investigate the functional role of ICCs in mediating phasic contractions, bladder strips were isolated from control and diabetic rats and mounted in tissue baths. • Strips were stimulated with low concentrations of the muscarinic receptor agonist carbachol (CCH; 0.1 µm) to induce phasic contractions and the effect of increasing concentrations (1-50 µm) of imatinib (Glivec® or Gleevec®, formerly STI571), a c-kit tyrosine kinase inhibitor, was then investigated. • For molecular studies, to detect expression of the c-kit tyrosine kinase receptor (c-kit), total cellular RNA was extracted from rat bladders and reverse-transcribed to obtain complementary DNA (cDNA). • Reverse transcription-polymerase chain reaction (RT-PCR) was then performed using primers specific to the c-kit sequence and amplified products separated by agarose gel electrophoresis. • Amplified PCR products were excised from the gel, sequenced and compared with the known c-kit sequence to confirm their identity. • For immunohistochemical detection, whole mount preparations of control rat bladders were fixed in acetone and labelled using antibodies directed to the ICC marker c-kit. RESULTS: • In functional studies, CCH induced phasic contractions in bladder strips from control and diabetic rats. Bladder strips from 1-week diabetic rats showed CCH-induced phasic contractions, which were greater in amplitude, but had lower frequency, than the controls, whilst no such differences were apparent at later time points of diabetes. • Imatinib decreased the amplitude and the frequency of the CCH-induced phasic contractions in both control and diabetic tissues in a concentration-dependent manner, although in diabetic tissues this effect was only seen at the higher concentrations of imatinib. RT-PCR of bladder cDNA yielded a single amplicon of 480 bp. • The sequence of this amplicon showed a 98% homology with the published c-kit sequence, thus confirming c-kit mRNA expression in both control and 1-week diabetic rat bladder. • Expression of c-kit protein was also detected in a network of cells on the edge of and between smooth muscle bundles of control rat bladders by positive immunoreactivity to c-kit specific antibodies. CONCLUSION: • These data show the presence of c-kit-positive ICCs in rat urinary bladder and their importance in mediating muscarinic receptor-induced phasic contractions of bladder strips from control and diabetic rats. The role of these ICCs does not seem to be significantly altered by the diabetic state.

The anxiolytic sertraline reduces the impact of psychological stress on bladder function in mice.

AIMS: To determine if treatment with the selective serotonin reuptake inhibitor (SSRI) sertraline reduces the bladder dysfunction caused by water avoidance stress in mice. MAIN METHODS: Adult female mice were randomly allocated to (1) Unstressed, (2) Stressed or (3) Stress + Sertraline experimental groups. Stressed mice were subjected to water avoidance for 1 h/day for 10 days and received sertraline or vehicle in drinking water, starting 10-days prior to the first stress exposure. Age matched control/unstressed mice were house under normal conditions without stress exposure. Voiding behaviour was assessed throughout the experimental protocol. After the final stress exposure, a blood sample was taken to measure plasma corticosterone levels and bladders were removed, catheterised and intravesical pressure responses recorded during distension and in response to pharmacological agents. KEY FINDINGS: Plasma corticosterone levels in sertraline-treated animals were equivalent to unstressed controls and significantly decreased compared to the stressed group. Voiding frequency was significantly increased in the stressed group, and treatment with sertraline significantly decreased voiding frequency, however, this remained elevated compared to unstressed control animals. Bladders from stressed mice displayed enhanced maximal contractile response to the muscarinic agonist carbachol and greater release of ACh in the serosal fluid, which was reduced to control levels by sertraline treatment. Spontaneous phasic contractions were not altered by stress but were significantly reduced in bladders from sertraline treated animals, relative to controls. SIGNIFICANCE: These results indicate that management of voiding dysfunction caused by psychological stress may be aided by the addition of an SSRI such as sertraline.

Bladder overactivity induced by psychological stress in female mice is associated with enhanced bladder contractility.

AIMS: To investigates the effects of water avoidance stress on voiding behaviour and functional bladder responses in mice. MAIN METHODS: Mice in the Stress group were exposed to water avoidance stress (WAS) for 1 h/day for 10 days, Controls were age-matched and housed normally. Voiding behaviour was measured periodically throughout the stress protocol and bladders were isolated 24-h after final stress exposure to measure bladder compliance, spontaneous phasic activity, contractile responses, and release of urothelial mediators. KEY FINDINGS: Repeated stress exposure induced a significant increase in plasma corticosterone levels in the WAS group compared to control. An overactive bladder phenotype was observed in WAS mice, causing a significant increase in the number of voiding events observed from as early as day-3, and a 7-fold increase following 10-days' stress. This increase in voiding frequency was associated with a significant decrease in void size, an increase in the number of small voids, but no change in total voided volume. Bladders from stressed mice showed a significant increase in the maximum responses to the muscarinic agonist carbachol (p < 0.01), in addition to enhanced pressure responses to the purinergic agonists ATP (p < 0.05) and αß-mATP (p < 0.05), and non-receptor mediated contractions to KCl (p < 0.05) compared to controls. Nerve-mediated bladder contractions to electric field stimulation were not significantly affected by stress, nor were spontaneous phasic contractions or release of urothelial ATP and acetylcholine. SIGNIFICANCE: Repeated exposure to water avoidance stress produced an overactive bladder phenotype, confirmed by increased voiding frequency, and associated with enhanced bladder contractile responses.

Psychological stress induced bladder overactivity in female mice is associated with enhanced afferent nerve activity.

Psychological stress has been linked to the development and exacerbation of overactive bladder symptoms, as well as afferent sensitisation in other organ systems. Therefore, we aimed to investigate the effects of water avoidance stress on bladder afferent nerve activity in response to bladder filling and pharmaceutical stimulation with carbachol and ATP in mice. Adult female C57BL/6J mice were exposed to either water avoidance stress (WAS) for 1 h/day for 10 days or normal housing conditions. Voiding behaviour was measured before starting and 24-h after final stress exposure and then animals were euthanised to measure afferent nerve activity in association with bladder compliance, spontaneous phasic activity, contractile responses, as well as release of urothelial mediators. WAS caused increased urinary frequency without affecting urine production. The afferent nerve activity at low bladder pressures (4-7 mmHg), relevant to normal physiological filling, was significantly increased after stress. Both low and high threshold nerves demonstrated enhanced activity at physiological bladder pressures. Urothelial ATP and acetylcholine release and bladder compliance were unaffected by stress as was the detrusor response to ATP (1 mM) and carbachol (1 µM). WAS caused enhanced activity of individual afferent nerve fibres in response bladder distension. The enhanced activity was seen in both low and high threshold nerves suggesting that stressed animals may experience enhanced bladder filling sensations at lower bladder volumes as well as increased pain sensations, both potentially contributing to the increased urinary frequency seen after stress.

Chronic psychological stress and lower urinary tract symptoms.

It is well established that lower urinary tract symptoms (LUTS), particularly urinary urgency and incontinence, cause stress and anxiety for patients. However, there is mounting evidence that the relationship between these two factors is bidirectional and that chronic psychological stress itself can result in the development of symptoms such as urinary frequency, urgency, incontinence, and pelvic pain. This review considers the evidence that such a relationship exists and reviews the literature from clinical and animal studies to identify some of the mechanisms that might be involved. Inflammatory responses induced by chronic stress appear to offer the strongest link to bladder dysfunction. There is overwhelming evidence, both in patients and animal models, for a release of pro-inflammatory cytokines and chemokines during periods of chronic stress. Furthermore, cytokines have been shown to cause bladder dysfunction and pain via actions in the central nervous system and locally in the bladder. In the brain and spinal cord, pro-inflammatory cytokines influence the regulation of micturition pathways by corticotropin-releasing factor (CRF) and its receptors, while peripherally cytokines affect bladder function, directly causing detrusor hypertrophy and afferent nerve hypersensitivity. There is little information on which treatments may have most benefit for stressed/anxious patients with LUTS, but animal studies suggest traditional drugs for overactive bladder (solifenacin, mirabegron) are more effective on LUTS than anxiolytic drugs (fluoxetine, imipramine). The preliminary preclinical data for CRF receptor antagonists is not consistent. A clearer understanding of the mechanisms involved in stress-induced LUTS should provide a basis for improved treatment of this condition.

The ß3-adrenoceptor mediates the inhibitory effects of ß-adrenoceptor agonists via the urothelium in pig bladder dome.

AIMS: Relaxation of detrusor muscle via ß-adrenoceptors may contribute to urine storage during bladder filling. Thus there is increasing interest in ß-adrenoceptor agonists as a potential treatment for detrusor overactivity. The role of the urothelium in bladder responses to ß-adrenoceptor agonists is not yet clear, although we have shown that these agonists have a greater inhibitory effect on detrusor contraction when the urothelium is intact. The aim was to determine which ß-adrenoceptor subtype is involved in this effect. METHODS: Paired strips of pig bladder dome mucosa-intact and mucosa-denuded, were mounted in tissue baths. Relaxation responses were obtained to ß-adrenoceptor agonists (isoprenaline, dobutamine, salbutamol or BRL37344) in carbachol pre-contracted tissues. Inhibitory effects were studied by obtaining concentration-response curves (CRCs) to carbachol in the presence and absence of ß-adrenoceptor agonists. The inhibitory effects of isoprenaline were also studied following incubation with ß-adrenoceptor antagonists (propranolol, CGP20712, ICI-118, 551 or SR 59230A; non selected, ß(1), ß(2) and ß(3) selective respectively). RESULTS: isoprenaline, dobutamine, salbutamol and BRL37344 all relaxed carbachol pre-contracted tissues and responses were similar in intact and denuded strips. In inhibition experiments, ß-adrenoceptor agonists caused rightward shifts of carbachol CRCs. In intact strips the shift was greater with isoprenaline and BRL37344, but not with dobutamine or salbutamol. This increased shift was still observed in tissues pre-incubated with propranolo, CGP20712 and ICI-118, 551, but not with SR 59230A. CONCLUSIONS: ß(3)-adrenoceptors are involved in mediating inhibitory effects of ß-adrenoceptor agonists on detrusor contractions via the urothelium in pig bladder dome.

Voiding Behavior and Efferent Bladder Function Altered in Mice Following Social Defeat but Not Witness Trauma.

Psychological stress is associated with bladder dysfunction, however, the local bladder mechanisms affected are not well understood. This study aimed to determine how psychological stress, caused by social defeat or witness trauma, affects voiding behavior and bladder function. Pairs of male C57Bl/6J mice were placed in a custom-made plexiglass chamber with an aggressor ARC(S) mouse for 1 h/day for 10 days. The social defeat mouse was in physical contact with the aggressor, while the witness was physically separated but could observe interactions between its cage-mate and the aggressor. Age matched control pairs were used for comparison. Voiding analysis was conducted periodically over the 10 days. An ex vivo whole bladder preparation was used to assess functional changes after the period of stress. Plasma corticosterone levels were significantly increased by both social defeat and witness trauma stress when compared to unstressed controls. Voiding analysis revealed a significant decrease in voiding frequency in the social defeat group compared to control animals, indicating an altered voiding phenotype. Witness trauma did not alter voiding behavior. Bladder contractile responses to cholinergic stimulation were not significantly altered in either stress group, nor was relaxation to the beta-adrenoceptor agonist isoprenaline. However, nerve evoked contractile responses were significantly increased at all frequencies in bladders from social defeat but not witness trauma mice. Purinergic contractile responses were also significantly enhanced in this group. Social defeat also resulted in increased urothelial acetylcholine release during bladder distension, with no change in ATP release. In conclusion, functional bladder changes are dependent upon stressor type. Enhanced urothelial acetylcholine may desensitize bladder sensory nerves, which, coupled with more efficient voiding contractions due to enhanced nerve-mediated and purinergic detrusor responses, may account for the altered voiding phenotype observed. This study reports a male model of social defeat stress with reduced urinary frequency, with no voiding changes observed in the witness.

Purinergic receptor mediated calcium signalling in urothelial cells.

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14-16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74-92%) responded to ATP (1 µM-1 mM), as indicted by an increase in intracellular calcium (iCa2+). PMUCs exhibited dose-dependent responses to ATP (10 nM-1 mM) in both calcium containing (2 mM, EC50 = 3.49 ± 0.77 µM) or calcium free (0 mM, EC50 = 9.5 ± 1.5 µM) buffers. However, maximum iCa2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X1-6, and P2Y1-2, P2Y4, P2Y6, P2Y11-14, but not P2X7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.

Spontaneous contractions of the pig urinary bladder: the effect of ATP-sensitive potassium channels and the role of the mucosa.

OBJECTIVE: To investigate the influence of the mucosa on the inhibitory effects of the ATP-sensitive potassium channel (K(ATP) channel) opener, cromakalim, on the spontaneous contractions of pig bladder strips from the bladder dome and trigone. Little is known about the influence of the mucosa on spontaneous contractions and whether the nature of these contractions differs between the bladder dome and trigone. MATERIALS AND METHODS: Paired longitudinal strips of female pig bladders were isolated from the dome and trigone. The mucosa was removed from one strip per pair and tissues were set up in organ baths. Spontaneous activity was allowed to develop and recorded, and then cumulative concentration-response curves to cromakalim were obtained. The time needed for spontaneous contractions to develop, the frequency and amplitude of spontaneous contractions, and the effect of cromakalim were analysed. The strips of mucosa removed from the dome to produce denuded strips were also analysed by immunofluorescence using antibodies specific for vimentin and alpha-smooth muscle actin (alpha-SMA). RESULTS: In the dome removal of the mucosa delayed the development of spontaneous contractions compared with mucosa-intact strips, whilst the trigone strips developed spontaneous contractions soon after set up in the organ baths irrespective of the presence or absence of mucosa. In the dome, cromakalim was more potent in suppressing spontaneous contractions when the mucosa was absent; whilst in the trigone the effects of cromakalim were similar in mucosa-intact and denuded strips. Upon examination of the strips of mucosa by immunofluorescence these strips were shown to contain cells positive for alpha-SMA or vimentin and cells positive for both, suggesting the presence of not only urothelium but also suburothelium and some detrusor smooth muscle bundles. CONCLUSION: In the dome, the urothelium and suburothelium reduce the inhibitory effect of cromakalim on spontaneous contractions, whilst in the trigone these structures appear to have little influence. The mechanism for generating spontaneous contractions in the intact strips seems to be linked to the urothelium and suburothelium in the dome but not in the trigone.

Effect of short-term androgen deficiency on bladder contractility and urothelial mediator release.

In men, testosterone levels decline by 1% per year after the age of 40. Reduced androgen levels may directly contribute to lower urinary tract symptoms and bladder dysfunction, although the mechanisms are unclear. This study examined the effect of low testosterone and testosterone replacement on key mechanisms involved in local bladder function. Intraluminal release of the mediators ATP and ACh in response to bladder distension was measured in whole bladders from rats 8 weeks following castration, whilst bladder contractility was assessed using isolated strips. Human urothelial cells were cultured under low, physiological and supra-physiological testosterone conditions for 24 h or 5 days, and stretch-induced release of ATP and ACh was measured. Phasic contractile activity of bladder strips, agonist-induced reponses to carbachol and isoprenaline and nerve-evoked contractions were unaffected by castration. The acetylcholinesterase inhibitor neostigmine significantly increased amplitude of phasic activity only in bladder strips following castration, and this was prevented by testosterone replacement. Intraluminal ACh release following bladder distension was significantly reduced following castration, whilst ATP release was unaffected. In contrast, stretch-induced ATP release from urothelial cells was significantly enhanced in low testosterone conditions, whilst ACh release was unaltered. Testosterone-replacement to physiological levels prevented these changes. Whilst androgen deficiency of 8 weeks does not directly affect contractility of bladder smooth muscle, urothelial mediator release is sensitive to changes in testosterone. These changes in mediator release may be an early effect of the decline in testosterone and could affect sensory pathways in the longer term, contributing to the urinary symptoms and bladder dysfunction seen in androgen-deficient men.