Release Of Urothelial Mediators Research Articles

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.

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.

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

AimsTo investigates the effects of water avoidance stress on voiding behaviour and functional bladder responses in mice. Main methodsMice 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 findingsRepeated 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 αβ-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. SignificanceRepeated exposure to water avoidance stress produced an overactive bladder phenotype, confirmed by increased voiding frequency, and associated with enhanced bladder contractile responses.

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, 24h after intraperitoneal treatment with cyclophosphamide (100mg/kg), ifosphamide (200mg/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.

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde's contribution to urothelial dysfunction in vitro.

The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10µM) than acrolein (100µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.

Elevated release of inflammatory but not sensory mediators from the urothelium is maintained following epirubicin treatment

Intravesical treatment of superficial bladder cancer with epirubicin is associated with local urological adverse effects, the causes of which are unknown. Our aim was to investigate the effects of epirubicin on the release of urothelial mediators and inflammatory cytokines. UROtsa cells were treated with epirubicin for 1 h at 37 °C. Release of urothelial transmitters and inflammatory cytokines was examined immediately, 24 h and 7 days following treatment. Release of ATP and nitric oxide were increased transiently after treatment with epirubicin (0.01 mg/ml), but this was not evident one-week after treatment. Basal prostaglandin E2 release was decreased at 24 h but not at 7 days. An increase in basal acetylcholine release and decrease in stretch-induced acetylcholine release were observed 24-h after treatment (0.01 mg/ml). One week following epirubicin treatment (0.001 mg/ml), stretch-induced, but not basal acetylcholine release, was observed. Secretion of interleukin-6 and interleukin-8 was increased 70-fold and 5-fold respectively, at 24 h (0.01 mg/ml). This was sustained and one week after epirubicin treatment (0.001 mg/ml), the increase in the secretion of both inflammatory cytokines was still evident.Epirubicin treatment induces several transient changes in urothelial function. However, the increased secretion of inflammatory cytokines (IL-6 and IL-8) is sustained and these mediators may be involved in the pathophysiology of bladder toxicity following intravesical epirubicin treatment.

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 2weeks of diabetes, although not at 12weeks. In contrast, intraluminal ACh release was unaltered by diabetes. Bladder compliance was also significantly enhanced at both 2 and 12weeks of diabetes, with greatly reduced intravesical pressures in response to distension. Nerve-evoked contractions of bladder strips were significantly greater at 2weeks of diabetes. When the urothelium was absent, nerve-evoked contractions were reduced, but contractions remained significantly elevated at lower frequencies of stimulation (<5Hz) 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.

NKA enhances bladder-afferent mechanosensitivity via urothelial and detrusor activation.

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

Ibuprofen Decreases Spontaneous Activity and Enhances Nerve-Evoked Contractions to Minimize Mitomycin C-Induced Bladder Dysfunction.

Inflammation may play a causal role in urological side effects reported following intravesical mitomycin C (MMC). Our aim was to investigate the effects of the nonsteroidal anti-inflammatory drug ibuprofen (IBU) on the cytotoxic potency of MMC and the potential for IBU to protect against bladder dysfunction. Malignant (RT4, T24) and normal (UROtsa) urothelial lines were treated with MMC followed by ibuprofen, with cell viability and caspase-3 activity assessed. Female C57BL/6JArc mice (Saline/Control, MMC, Saline + IBU, and MMC + IBU) received intravesical treatment (1 hour) with saline or MMC (2 mg/ml), with IBU (1 mg/ml) delivered in drinking water (for 7 days). Voiding pattern analysis was conducted prior to and following (1, 3, 7 days) treatment. A whole-bladder preparation was used to assess compliance, contractile responses, and urothelial-mediator release. Ibuprofen selectively increased the cytotoxic potency of MMC and caspase-3 activity in both malignant cells lines but not in UROtsa. MMC significantly increased voiding frequency at 24 hours and 3 days, whereas administration of ibuprofen significantly reduced this effect. MMC significantly increased the frequency of spontaneous contractions from 2.3 ± 0.5 contractions/min in saline controls to 4.8 ± 0.16 contractions/min, with ibuprofen protecting against this change. Interestingly, although nerve-evoked responses were not altered by MMC, they were increased in both IBU groups. Ibuprofen improved voiding dysfunction following MMC treatment by reducing spontaneous phasic activity and enhancing nerve-mediated contractions. Ibuprofen use in bladder cancer patients may help to minimize the urological adverse effects associated with intravesical MMC.

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 8weeks following castration, whilst bladder contractility was assessed using isolated strips. Human urothelial cells were cultured under low, physiological and supra-physiological testosterone conditions for 24h or 5days, 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 8weeks 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.

Urothelial purine release during filling of murine and primate bladders.

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

Recovery of urothelial mediator release but prolonged elevations in interleukin-8 and nitric oxide secretion following mitomycin C treatment.

Intravesical mitomycin C (MMC) is commonly used to treat bladder cancer but is associated with local adverse effects. Here, we investigate the effects of MMC on release of urothelial mediators and production of inflammatory cytokines. Recovery and the effects of repeat treatment were also investigated. Urothelial cells were treated with MMC for 2h at 37°C. Immediately, 24h and 7days following MMC treatment, effects were assessed in terms of changes in ATP, acetylcholine and PGE2 release, and the presence of inflammatory cytokines and nitric oxide in incubation medium. Endpoints were also assessed 7days after repeat MMC treatment. Immediately following MMC treatment at the clinical concentration (2mg/mL), stretch-induced ATP release was significantly decreased, basal acetylcholine release was enhanced, while basal PGE2 was depressed and response to stretch increased. Twenty-four hours after treatment, basal acetylcholine was significantly increased ([MMC] ≥0.0002mg/mL) and basal PGE2 enhanced (0.02mg/mL). A 326-fold increase in interleukin-8 secretion and significant increase in nitric oxide release were also detected at 24h. One week after single and repeat MMC treatment, urothelial ATP, acetylcholine and PGE2 had recovered while the increase in nitric oxide and interleukin-8 was still evident. These results indicate that urothelial mediator release is affected by MMC treatment with full recovery by 1week. However, a concurrent increase in secretion of the inflammatory cytokine interleukin-8 and nitric oxide was also detected 24h following treatment, which were still evident after the recovery period. These changes may play a role in the local adverse effects associated with intravesical MMC treatment.

Alterations in acetylcholine, PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide

The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1–100μM) or LPS (1–100ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100μM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100μM), stretch-induced release for pseudomonal LPS (⩾10ng/mL) and both basal and stimulated release for E. coli LPS (⩾10ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100μM) or LPS (100ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.

Induction of inflammatory cytokines and alteration of urothelial ATP, acetylcholine and prostaglandin E2 release by doxorubicin

Intravesical treatment with cytotoxic drugs such as doxorubicin is associated with local adverse effects in bladder cancer patients. Here we investigate the effects of doxorubicin on urothelial release of ATP, acetylcholine and prostaglandin E2, and production of inflammatory cytokines. Urothelial cells were treated with doxorubicin for 1h at 37°C. Immediately or 24h following treatment the level of ATP, acetylcholine and prostaglandin E2 released under basal and stimulated conditions was measured and compared to release from vehicle treated control cultures. The presence of inflammatory cytokines, in culture medium was also assessed 24h after doxorubicin pre-treatment. Immediately following treatment, stimulated ATP release was inhibited at doxorubicin concentrations ≥1μg/ml and showed partial recovery at 24h. Immediately following treatment, basal acetylcholine release was increased by doxorubicin at its clinical concentration (1mg/ml), while a concentration-dependent decrease in stimulated acetylcholine release was observed. Twenty four hour after treatment, basal acetylcholine release was increased in culture treated with 0.01mg/ml doxorubicin while stimulated acetylcholine release remained depressed. A significant increase in prostaglandin E2 release was observed in cells immediately and 24h after treatment with doxorubicin. A 5.5- and 2-fold increase in interleukin -8 and -1β secretion, respectively was detected 24h following doxorubicin treatment. These findings indicate that inflammatory cytokines interleukin-8 and -1β are induced and urothelial mediator release is affected by treatment with doxorubicin at clinically relevant concentrations and durations of treatment. These changes may play a role in the adverse effects associated with intravesical doxorubicin treatment.