Inhibition of PDE-4 isoenzyme attenuates frequency and overall contractility of agonist-evoked ureteral phasic contractions.

The aim of this study was to investigate the functional role of phosphodiesterase enzymes (PDE) in the isolated porcine ureter. Distal ureteral strips were mounted in organ baths and pre-contracted with 5-HT (100 µM). Upon generation of stable phasic contractions, PDE-4 and PDE-5 inhibitors were added cumulatively to separate tissues. PDE-4 inhibitors, such as rolipram (10 nM and greater) and roflumilast (100 nM and greater), resulted in significant attenuation of ureteral contractile responses, while a higher concentration of piclamilast (1 µM and greater) was required to induce a significant depressant effect. The attenuation effect by rolipram was abolished by SQ22536 (100 µM). PDE-5 inhibitors, such as sildenafil and tadalafil, were not nearly as effective and were only able to suppress the 5-HT-induced contractions at higher concentrations of 1 µM. Rolipram significantly enhanced the depressant effect of forskolin, while sodium nitroprusside-induced attenuation of contractile responses remained unchanged in the presence of tadalafil. In summary, our study demonstrates that PDE-4 inhibitors are effective in attenuating 5-HT-induced contractility in porcine distal ureteral tissues, while PDE-5 inhibitors are less effective. These findings suggest that PDE-4 inhibitors, such as rolipram, may hold promise as potential therapeutic agents for the treatment of ureteral disorders attributable to increased intra-ureteral pressure.

Phosphodiesterase-5 inhibitors tadalafil and sildenafil potentiate nitrergic-nerve mediated relaxations in the bladder vasculature.

Recent studies suggest that lower urinary tract dysfunction may arise due to changes in local perfusion. Phosphodiesterase-5 inhibitors can improve urinary bladder blood flow, although the local mechanisms have not been fully elucidated. The aim was to pharmacologically characterise the vascular supply to the bladder and determine the mechanisms underlying the effects of the phosphodiesterase-5 inhibitors tadalafil and sildenafil. Responses of isolated rings of porcine superior vesical arteries to electrical field stimulation (EFS) were measured in the absence and presence of inhibitors of key neurotransmitter systems. Vasodilation responses to nitric oxide (NO) donors were also recorded, and the effects of phosphodiesterase-5 inhibitors on all responses determined. EFS caused biphasic responses with an initial vasoconstriction and a slower developing vasodilation. Vasoconstriction was mediated by ATP (55%) and noradrenaline (45%) release, whilst vasodilation was reduced by L-NNA (100 µM) (80%) and propranolol (1 µM) (20%). The nitrergic component was inhibited (81%) by L-NPA, a selective inhibitor of neuronal nitric oxide synthase (nNOS). Endothelial removal did not affect vasodilation. Tadalafil and sildenafil depressed noradrenaline-evoked vasoconstriction (by 26.8% and 35.5% respectively, P < 0.01), enhanced vasodilation to EFS (by 27.8% and 51.8% respectively, p < 0.01) and enhanced responses to NO donors nitroprusside, SIN-1, and SNAP, increasing pIC50 values (P < 0.01), without affecting maximal responses. In conclusion, neuronal NOS has a predominant role in regulating vascular tone of the porcine superior vesical artery and potentiation of nNO-mediated vasodilation is the primary mechanism underlying effects of phosphodiesterase-5 inhibitors in the bladder vasculature.

Ageing influences detrusor contractions to prostaglandin, angiotensin, histamine and 5-HT (serotonin), independent to the Rho kinase and extracellular calcium pathways.

Ageing is associated with deteriorating urinary bladder function and an increasing prevalence of disorders such as underactive bladder. There are suggestions that G protein-coupled receptor (GPCR) second messenger pathways are altered during ageing, rather than the receptor proteins themselves. The aim of this study was to identify age-related variations in GPCR activation systems in urinary bladder smooth muscle (detrusor). Isolated porcine detrusor strips were mounted in organ baths and contractile responses induced by receptor agonists were assessed and compared between juvenile (6 months) and adult (2 years) animals. The effects of drugs disrupting intracellular calcium signalling were also studied. Adult tissue was far more sensitive to stimulation by 5-hydroxytryptamine (42% greater increase than juvenile), prostaglandin-E2 (26% greater increase), and angiotensin-II (39% greater increase), however less sensitive to histamine. Although nifedipine and Y-27632 impacted the contraction to all agonists, there were no significant differences between juvenile and adult detrusor. Impairment of IP3-mediated calcium release by 2-aminoethyl diphenylborinate had no effect on any contractile activity, except for neurokinin-A which inhibited both juvenile and adult detrusor, and prostaglandin-E2 which inhibited juvenile. Carbachol, histamine, 5-hydroxytryptamine, and angiotensin-II were not affected by the application of 2-aminoethyl diphenylborinate. In conclusion, the contractile responses to all the GPCR agonists involved extracellular calcium influx and calcium sensitisation, but for prostaglandin-E2 the dependence on calcium from intracellular sources was greater in the younger animals.

Antimuscarinic actions on bladder urothelium and lamina propria contractions are similar to those observed in detrusor smooth muscle preparations.

OBJECTIVES: Antimuscarinic medications are the first-line treatments for overactive bladder, the most common form of bladder dysfunction. Their primary action is thought to block detrusor muscarinic receptors. It is unclear, however, if these therapeutics have actions on other tissues within the lower urinary tract. This study assessed whether clinical antimuscarinics have a functional impact on urothelium with lamina propria (U&LP) tissue. METHODS: Strips of porcine detrusor and U&LP were mounted in carbogen-gassed Krebs-bicarbonate solution at 37°C. The tissues were paired with carbachol-response curves performed in the absence or presence of each antimuscarinic. pEC50 values for each curve were analyzed and estimated affinities calculated. RESULTS: Both detrusor and U&LP tissues contracted with muscarinic receptor stimulation, which was inhibited by commonly used antimuscarinics. In detrusor samples (p < 0.001 for all), right parallel shifts from the control were observed in response to oxybutynin (1 µM), solifenacin (1 µM), tolterodine (1 µM), darifenacin (100 nM), trospium (100 nM) and fesoterodine (100 nM). This shift was consistent in U&LP samples, with no significant differences between the two layers. CONCLUSIONS: The data suggests that clinical antimuscarinics are as effective at inhibiting tonic contractions of the U&LP as they are on detrusor, presenting the U&LP as an alternate target of these medications in the treatment of lower urinary tract symptoms.

The role of intracellular calcium and Rho kinase pathways in G protein-coupled receptor-mediated contractions of urinary bladder urothelium and lamina propria.

The influence of extracellular and intracellular calcium on smooth muscle contractile activity varies between organs. In response to G protein-coupled receptor (GPCR) stimulation, the urinary bladder detrusor muscle has shown a 70% dependence on extracellular calcium, whereas the urothelium and lamina propria (U&LP) has a 20%-50% dependence. However, as this only accounts for partial contractile activity, the contribution of intracellular calcium and calcium sensitization pathways remains unclear. This study assessed the role of intracellular signaling pathways on GPCR-mediated urinary bladder U&LP contraction. Porcine U&LP responses to activation of the Gq/11-coupled muscarinic, histamine, 5-hydroxytryptamine (serotonin), neurokinin, prostaglandin, and angiotensin II receptors were assessed with three selective inhibitors of store-released intracellular calcium, 2-aminoethyl diphenylborinate (2-APB), cyclopiazonic acid (CPA), and ruthenium red, and three Rho kinase inhibitors, fasudil, Y-27632, and GSK269962. There was no discernible impact on receptor agonist-induced contractions of the U&LP after blocking intracellular calcium pathways, suggesting that this tissue is more sensitive to alterations in the availability of extracellular calcium. However, an alternative mechanism of action for GPCR-mediated contraction was identified to be the activation of Rho kinase, such as when Y-27632 significantly reduced the GPCR-mediated contractile activity of the U&LP by approximately 50% (P < 0.05, n = 8). This suggests that contractile responses of the bladder U&LP do not involve a significant release of calcium from intracellular stores, but that Gq/11-coupled receptor activation causes calcium sensitization via Rho kinase. This study highlights a key role for Rho kinase in the urinary bladder, which may provide a novel target in the future pharmaceutical management of bladder contractile disorders.

Drivers of Radioresistance in Prostate Cancer.

Prostate cancer (PCa) is the second most commonly diagnosed cancer worldwide. Radiotherapy remains one of the first-line treatments in localised disease and may be used as monotherapy or in combination with other treatments such as androgen deprivation therapy or radical prostatectomy. Despite advancements in delivery methods and techniques, radiotherapy has been unable to totally overcome radioresistance resulting in treatment failure or recurrence of previously treated PCa. Various factors have been linked to the development of tumour radioresistance including abnormal tumour vasculature, oxygen depletion, glucose and energy deprivation, changes in gene expression and proteome alterations. Understanding the biological mechanisms behind radioresistance is essential in the development of therapies that are able to produce both initial and sustained response to radiotherapy. This review will investigate the different biological mechanisms utilised by PCa tumours to drive radioresistance.

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 Dependence of Urinary Bladder Responses on Extracellular Calcium Varies Between Muscarinic, Histamine, 5-HT (Serotonin), Neurokinin, Prostaglandin, and Angiotensin Receptor Activation.

With many common bladder diseases arising due to abnormal contractions, a greater understanding of the receptor systems involved may aid the development of future treatments. The aim of this study was to identify any difference in the involvement of extracellular calcium (Ca2+) across prominent contractile-mediating receptors within cells lining the bladder. Strips of porcine urothelium and lamina propria were isolated from the urinary bladder dome and mounted in isolated tissue baths containing Krebs-bicarbonate solution, perfused with carbogen gas at 37°C. Tissue contractions, as well as changes to the frequency and amplitude of spontaneous activity were recorded after the addition of muscarinic, histamine, 5-hydroxytryptamine, neurokinin-A, prostaglandin E2, and angiotensin II receptor agonists in the absence and presence of 1 µM nifedipine or nominally zero Ca2+ solution. The absence of extracellular Ca2+ influx after immersion into nominally zero Ca2+ solution, or the addition of nifedipine, significantly inhibited the contractile responses (p < 0.05 for all) after stimulation with carbachol (1 µM), histamine (100 µM), 5-hydroxytryptamine (100 µM), neurokinin-A (300 nM), prostaglandin E2 (10 µM), and angiotensin II (100 nM). On average, Ca2+ influx from extracellular sources was responsible for between 20-50% of receptor-mediated contractions. This suggests that although the specific requirement of Ca2+ on contractile responses varies depending on the receptor, extracellular Ca2+ plays a key role in mediating G protein-coupled receptor contractions of the urothelium and lamina propria.

Mirabegron attenuates porcine ureteral contractility via α1-adrenoceptor antagonism.

The ß3-agonist mirabegron is thought to induce relaxation of the detrusor muscle, contributing to the improvement of overactive bladder symptoms. There has been recent interest in purposing mirabegron as a medical expulsive therapy drug to improve the passage of smaller kidney stones by relaxing the ureteral smooth muscles. The aim of this study was to determine the effects of mirabegron on the activity of the ureter. Additionally, we investigated the receptor and mechanisms through which mirabegron exerts these effects. In vitro agonist-induced responses of isolated porcine distal ureteral tissues were measured in the absence and presence of mirabegron in organ bath experiments. The responses were expressed as frequency, area under the curve and maximum amplitude. Mirabegron at concentrations of 100 nM and lower failed to suppress phenylephrine- or 5-HT-induced contractions in the porcine ureteral strip. Mirabegron at 1 µM and 10 µM produced a rightward shift of phenylephrine concentration-response curves in these tissues. This effect of mirabegron (10 µM) was not present in 5-HT concentration-response curves. The mirabegron effect on phenylephrine-induced contractions was also not abolished by ß-adrenoceptor antagonist SR 59230A (10 µM), ß-adrenoceptor antagonist propranolol (10 µM), α2-adrenoceptor antagonist yohimbine (30 nM), and nitric oxide synthase inhibitor L-NNA (10 µM). The present results show that mirabegron suppresses ureteral contractile responses in the porcine ureter via α1-adrenoceptor antagonism, since their effects were not present when the tissues were contracted with 5-HT. Furthermore, the inhibitory effects by mirabegron were not affected by ß3-adrenoceptor antagonists.

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.

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.

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 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.

Racemic synephrine found in Citrus aurantium-listing pre-workout supplements suggests a non-plant-based origin.

Multi-ingredient pre-workout supplements (MIPS) contain Citrus aurantium as a source of bioactive amines such as p-synephrine, but concerns regarding the authenticity of ingredients in some supplements as well as adverse effects from consumption have been raised. R-(-)-Synephrine is the predominant enantiomer in Citrus aurantium extracts while synthetic preparations are often racemic. The aims of this study were to develop a screening method to determine the ratio of synephrine enantiomers in pre-workout supplements listing Citrus aurantium and to assess the ingredient authenticity by directly comparing their ratios to that found in Citrus aurantium standardised reference materials (SRMs). Quantification of enantiomers in the supplements and SRMs was achieved using a validated, high-performance liquid chromatography-single quadrupole mass spectrometry (HPLC-UV-QDa) direct enantioseparation method with a cellobiohydrolase (CBH) column (100 × 4.0 mm, 5 µM) and UV detection at 225 nm. Citrus aurantium SRMs were found to have an average enantiomeric ratio of 94:6 (R:S) with total synephrine ranging from 5.7 to 90.2 mg/g. Within the pilot sample of pre-workout supplements tested, only 42% (5/12) had enantiomeric ratios consistent with the SRMs with total synephrine ranging from 0.03 to 91.2 mg/g. For the remaining supplements, four had racemic ratios of synephrine (0.14 to 5.4 mg/g), two lacked any detectable levels of synephrine, and one had solely the S-(+)-enantiomer (0.15 mg/g). These results bring the authenticity of labelling of some pre-workout supplements into question and highlight the need for more stringent labelling regulations and testing for dietary supplements.

Renal artery responses to trace amines: Multiple and differential mechanisms of action.

PURPOSE: The rise in consumption of dietary supplements containing the trace amines p-tyramine, p-synephrine and p-octopamine has been associated with cardiovascular side effects. Since renal blood flow plays an important role in blood pressure regulation, this study investigated the mechanisms of action of these trace amines on isolated porcine renal arteries. MAIN METHODS: Contractile responses to amines were investigated in noradrenaline-depleted rings of porcine main renal arteries in the absence and presence of the α1-adrenoceptor antagonist, prazosin (1 µM), ß-adrenoceptor antagonist, propranolol (1 µM), or the trace amine-associated receptor (TAAR-1) antagonist, EPPTB (RO-5212773; 100 nM or 100 µM). KEY FINDINGS: All three amines induced constrictor responses of similar magnitude and potency. However, their mechanisms of action on the renal artery appeared to differ. Depleting endogenous noradrenaline stores significantly reduced maximum responses to tyramine and synephrine, but less for octopamine. When direct responses were examined after depleting tissues of noradrenaline, responses to synephrine and octopamine, but not tyramine, were reduced in the presence of prazosin(1 µM) and potentiated in the presence of propranolol (1 µM) or L-NNA (100 µM). Generally, vasoconstrictor responses remaining after noradrenaline-depletion and α-adrenoceptor blockade were not affected by the TAAR-1 antagonist EPPTB (0.1-100 µM), although responses to low concentration of synephrine and octopamine were enhanced by this antagonist. SIGNIFICANCE: Tyramine appears to mediate constriction of the renal artery mainly via an indirect sympathomimetic mechanism, whereas synephrine and octopamine exert additional direct effects on α1-adrenoceptors and possibly contractile TAAR (not TAAR-1). The two amines also activate simultaneous inhibitory responses via ß-adrenoceptors, TAAR-1 and nitric oxide release.

17ß-estradiol and ureteral contractility: A role for the G protein-coupled estrogen receptor.

The aim of this study was to investigate the unknown effects of 17ß-estradiol (E2) on ureteral contractility and the receptor and mechanisms involved. By utilising isolated porcine distal ureteral strips, we observed that E2 (30-300 µM) and a G protein-coupled estrogen receptor specific agonist G-1 (30 µM) both increased the frequency of phasic contractions of the ureter (P<0.05). E2 also decreased the maximum amplitude of these contractions (P<0.05). The G protein-coupled estrogen receptor specific antagonist G-36 (10 µM) reversed E2 enhancement effects on frequency, but did not alter its effects on maximum amplitude of contractile responses. Additionally, it was observed that the effects of E2 were unaltered by removing the urothelium, inhibiting nitric oxide and prostaglandin production or preventing neuronal conduction. In the presence of a potassium channel blocker, 4-aminopyridine (10 µM), the effects of E2 on frequency were prevented. This finding suggests that G protein-coupled estrogen receptor mediates the increase in frequency of ureteral phasic contractions induced by E2 via activation of potassium channels, while E2 alters the amplitude of these contractions through an unknown mechanism.

Comparative effects of angiotensin II on the contractility of muscularis mucosae and detrusor in the pig urinary bladder.

To explore contractile actions of angiotensin II (ATII) on the muscularis mucosae (MM) of the bladder, ATII-induced contractions were compared between MM and the detrusor smooth muscle (DSM) of the pig bladder by isometric tension recordings. Effects of ATII on spontaneous Ca2+ transients in MM were visualized using Cal-520 fluorescence. ATII receptor type 1 (ATR1) expression in MM and DSM was also examined by immunohistochemistry. ATII (1 nM-1 µM) caused phasic contractions of MM in a concentration-dependent manner, while ATII (10 nM-10 µM) had no or marginal effects on DSM contractility. ATII (100 nM)-induced MM contractions had an amplitude of approximately 70% of carbachol (1 µM)-induced or 90% of U46619 (100 nM)-induced contractions. Candesartan (10 nM), an ATR1 blocker, prevented the contractile effects of ATII (1 nM) in MM, while ATR1 immunofluorescence was greater in MM than DSM. ATII (10-100 pM) increased the frequency but not the amplitude of spontaneous Ca2+ transients in MM. Both urothelium-intact and -denuded MM strips developed comparable spontaneous phasic contractions, but ATII, carbachol and U46619-induced contractions were significantly larger in urothelium-denuded than urothelium-intact MM strips. In conclusion, the MM appears to have a much greater sensitivity to ATII compared with DSM that could well sense circulating ATII, suggesting that MM may be the predominant target of contractile actions induced by ATII in the bladder while the urothelium appears to inhibit MM contractility.

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.