Urothelial ATP Release Research Articles

Control of nerve-mediated and urothelial ATP release by a protein kinase G-dependent pathway in the mouse bladder

Aim:ATP signalling is involved in urinary bladder motor and sensory pathways, including stimulation-mediated release from parasympathetic varicosities to detrusor muscle and from urothelial cells when mechanically stressed. Both modalities are present in humans and other mammals but are especially prominent in overactive bladder syndromes. There is therefore an unmet need to understand how to regulate such release. This study tested the hypothesis that the nitric oxide (NO•)/ soluble guanylate cyclase (sGC)/ cyclic GMP/ protein kinase-G (PKG) pathway has a central role. Methods:In vitro nerve-mediated contractions and ATP/acetylcholine (ACh) release were measured from bladder wall strips, as was ATP release from urothelial cell suspensions subject to mechanical stresses. Enhanced spontaneous contractile activity was also measured in bladder wall preparations of spinal cord-injured mice. Interventions were designed to increase cellular cGMP levels (a cell-permeable cGMP analogue, a NO•donor, a phosphodiesterase inhibitor (PDEI), a sGC activator), or agents to reduce activity of pathway enzymes (sCG or PKG). Results:ATP-dependent contractions were reduced by the above interventions, as was ATP release; but ACh-dependent contractions and ACh release were unaffected. Spontaneous contractile activity was also reduced by the cGMP analogue and by a PDEI. ATP release from urothelial cell suspensions was also reduced by similar interventions. Conclusions:ATP release from efferent nerves and from urothelial cells were selectively reduced by upregulating the NO•/sGC/cGMP/PKG pathway. Translational aspects are discussed with respect to purinergic pathways and overactive bladder pathologies.

Activation of Piezo1 channels enhances spontaneous contractions of isolated human bladder strips via acetylcholine release from the mucosa

Enhanced spontaneous bladder contractions (SBCs) have been thought one of the important underlying mechanisms for detrusor overactivity (DO). Piezo1 channel has been demonstrated involved in bladder function and dysfunction in rodents. We aimed to investigate the modulating role of Piezo1 in SBCs activity of human bladder. Human bladder tissues were obtained from 24 organ donors. SBCs of isolated bladder strips were recorded in organ bath. Piezo1 expression was examined with reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining. ATP and acetylcholine release in cultured human urothelial cells was measured. Piezo1 is abundantly expressed in the bladder mucosa. Activation of Piezo1 with its specific agonist Yoda1 (100 nM–100 μM) enhanced the SBCs activity in isolated human bladder strips in a concentration-dependent manner. The effect of Yoda1 mimicked the effect of a low concentration (30 nM) of carbachol, which can be attenuated by removing the mucosa, blocking muscarinic receptors with atropine (1 μM), and blocking purinergic receptors with pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS, 30 μM), but not by tetrodotoxin (1 μM). Activation of urothelial Piezo1 with Yoda1 (30 μM) or hypotonic solution induced the release of ATP and acetylcholine in cultured human urothelial cells. In patients with benign prostatic hyperplasia, greater Piezo1 expression was observed in bladder mucosa from patients with DO than patients without DO. We conclude that upregulation and activation of Piezo1 may contribute to DO generation in patients with bladder outlet obstruction by promoting the urothelial release of ATP and acetylcholine. Inhibition of Piezo1 may be a novel therapeutic approach in the treatment of overactive bladder.

The Pannexin 1 Channel and the P2X7 Receptor Are in Complex Interplay to Regulate the Release of Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

The bladder urothelium releases ATP into the lamina propria (LP) during filling, which can activate P2X receptors on afferent neurons and trigger the micturition reflex. Effective ATP concentrations are largely dependent on metabolism by membrane-bound and soluble ectonucleotidases (s-ENTDs), and the latter are released in the LP in a mechanosensitive manner. Pannexin 1 (PANX1) channel and P2X7 receptor (P2X7R) participate in urothelial ATP release and are physically and functionally coupled, hence we investigated whether they modulate s-ENTDs release. Using ultrasensitive HPLC-FLD, we evaluated the degradation of 1,N6-etheno-ATP (eATP, substrate) to eADP, eAMP, and e-adenosine (e-ADO) in extraluminal solutions that were in contact with the LP of mouse detrusor-free bladders during filling prior to substrate addition, as an indirect measure of s-ENDTS release. Deletion of Panx1 increased the distention-induced, but not the spontaneous, release of s-ENTDs, whereas activation of P2X7R by BzATP or high concentration of ATP in WT bladders increased both. In Panx1-/- bladders or WT bladders treated with the PANX1 inhibitory peptide 10Panx, however, BzATP had no effect on s-ENTDS release, suggesting that P2X7R activity depends on PANX1 channel opening. We concluded, therefore, that P2X7R and PANX1 are in complex interaction to regulate s-ENTDs release and maintain suitable ATP concentrations in the LP. Thus, while stretch-activated PANX1 hinders s-ENTDS release possibly to preserve effective ATP concentration at the end of bladder filling, P2X7R activation, presumably in cystitis, would facilitate s-ENTDs-mediated ATP degradation to counteract excessive bladder excitability.

In Vivo Luminal Measurement of Distension-evoked Urothelial ATP Release in Rodents.

ATP, released from the urothelium in response to bladder distension, is thought to play a significant sensory role in the control of micturition. Therefore, accurate measurement of urothelial ATP release in a physiological setting is an important first step in studying the mechanisms that control purinergic signaling in the urinary bladder. Existing techniques to study mechanically evoked urothelial ATP release utilize cultured cells plated on flexible supports or bladder tissue pinned into Ussing chambers; however, each of these techniques does not fully emulate conditions in the intact bladder. Therefore, an experimental setup was developed to directly measure ATP concentrations in the lumen of the rodent urinary bladder. In this setup, the bladders of anesthetized rodents are perfused through catheters in both the dome of the bladder and via the external urethral orifice. Pressure in the bladder is increased by capping the urethral catheter while perfusing sterile fluid into the bladder through the dome. Measurement of intravesical pressure is achieved using a pressure transducer attached to the bladder dome catheter, akin to the setup used for cystometry. Once the desired pressure is reached, the urethral catheter's cap is removed, and fluid collected for ATP quantification by luciferin-luciferase assay. Through this experimental setup, the mechanisms controlling both mechanical and chemical stimulation of urothelial ATP release can be interrogated by including various agonists or antagonists into the perfusate or by comparing results between wildtype and genetically modified animals.

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.

PD06-10 URINARY ATP LEVELS: A POTENTIAL BIOMARKER OF OVERACTIVE BLADDER TREATMENT RESPONSE FOLLOWING ONABOTULINUM TOXIN-A INJECTIONS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Female Incontinence: Therapy I (PD06)1 Sep 2021PD06-10 URINARY ATP LEVELS: A POTENTIAL BIOMARKER OF OVERACTIVE BLADDER TREATMENT RESPONSE FOLLOWING ONABOTULINUM TOXIN-A INJECTIONS Samantha Freeman, Laura Tellechea, Ilir Agalliu, Melissa Laudano, Nitya Abraham, and Sylvia Suadicani Samantha FreemanSamantha Freeman More articles by this author , Laura TellecheaLaura Tellechea More articles by this author , Ilir AgalliuIlir Agalliu More articles by this author , Melissa LaudanoMelissa Laudano More articles by this author , Nitya AbrahamNitya Abraham More articles by this author , and Sylvia SuadicaniSylvia Suadicani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001974.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Overactive bladder (OAB) is diagnosed clinically based on symptoms. However, a biomarker may help provide objective measurement of disease severity and improvement after treatment. ATP is released from the urothelium and levels tend to be higher in animal models and patients with OAB. Bladder onabotulinum toxin-A (BTX-A) injections decrease stretch-induced ATP release in animal models, yet little is known about the effect of BTX-A on human urine ATP levels. The objective of this study is to quantify changes in voided urine ATP levels and correlate with OAB-V8 symptom improvement in women treated with BTX-A injection. METHODS: Urine samples were collected and frozen on dry ice from women prior to and after 100 units of BTX-A treatment. Urine ATP levels were quantified using the luciferin-luciferase luminescent assay. OAB-V8 questionnaires were administered at time of urine collection. Women with positive urine cultures or neurogenic bladder were excluded. Degree of OAB-V8 score improvement was used to categorize participants: non-responders (change<8), mild responders (change 8-16), moderate responders (change 17-24), and marked responders (change>24). Percent change in ATP level was compared based on OAB-V8 score improvement and statistical significance was determined using the Mann-Whitney test. RESULTS: Urine was collected from 23 female patients (OAB-V8 scores 2 to 40); 7 were excluded. Median age was 53.5 years (49.5-61.5). 8 patients had chronic pain disorders, 2 patients had Type 2 diabetes, and 6 patients had prior pelvic surgery. After BTX-A injections, responders had significantly lower median ATP levels compared to non-responders at 2 weeks (p=0.0268). When patients were classified according to OAB-V8 score improvement (non-responder, mild, moderate, marked responders), the improvement directly correlated with a decrease in ATP levels (Figure 1). CONCLUSIONS: Decreases in urine ATP levels after BTX-A injections were associated with clinical improvement in OAB symptoms. This may be the result of a direct or indirect effect of BTX-A on mechanisms of urothelial ATP release and signaling. Quantification of urine ATP may provide a valuable tool to assess treatment response and guide further management. Source of Funding: N/A © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e103-e104 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Samantha Freeman More articles by this author Laura Tellechea More articles by this author Ilir Agalliu More articles by this author Melissa Laudano More articles by this author Nitya Abraham More articles by this author Sylvia Suadicani More articles by this author Expand All Advertisement Loading ...

The nitric oxide-cyclic guanosine monophosphate pathway inhibits the bladder ATP release in response to a physiological or pathological stimulus.

The release of ATP from the epithelium of the urinary bladder (urothelium) in response to mechanical/chemical stimuli contributes to the visceral sensation in the micturition reflex. The nitric oxide (NO)‐mediated induction of cyclic guanosine monophosphate (cGMP) has been detected in urothelial cells and may inhibit the micturition reflex. However, the function of the NO‐cGMP pathway in the regulation of urothelial ATP release remains poorly understood in contrast to its effects on smooth muscles or primary afferent nerves. Therefore, we investigated the relevance of the NO‐cGMP pathway to ATP release on the mucosal side in the present study. The administration of l‐arginine (NO precursor) or NOC 12 (NO donor) significantly reduced ATP release to the mucosal side at a physiologically normal urine storage pressure (5 cmH2O). L‐NAME (NO synthase inhibitor) significantly increased the distention‐induced release of ATP. The phosphodiesterase‐5 inhibitor, sildenafil, which increases cGMP levels, inhibited distention‐induced ATP release. Furthermore, sildenafil significantly reduced ATP release in response to the administration of lipopolysaccharide. These results suggest that the NO‐cGMP pathway inhibited urothelial ATP release during the storage phase under both physiological and pathological conditions.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase.

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.

MP54-06 PANNEXIN 1 CHANNELS INVOLVEMENT IN MECHANISM OF STRESS-INDUCED PELVIC PAIN AND URINARY SYMPTOMS IN MICE

INTRODUCTION AND OBJECTIVE: Pannexin 1 (Panx1) channels are key components of the urothelial mechanosensory and mechanotransduction system. They are mechanosensitive and provide a direct conduit for urothelial ATP release in response to bladder distension. Little is known of the role of Panx1 channels in bladder pathology. The goal of this study is to evaluate if Panx1 channels are involved in emergence of pelvic pain and urinary symptoms in an animal model of stress. METHODS: Wildtype (WT) and Panx1-null mice, and the stress model of 96 hours of continuous illumination (CI) were used in this study. Pelvic tactile sensitivity and bladder function were evaluated at baseline (pre-CI) and the day after the 96 hours of CI using the Von Frey test and the void stain on paper (VSOP) method, respectively. ATP levels in spontaneously voided urine were quantified pre- and post-CI using the luciferin-luciferase assay. Bladders were harvested from CI stressed and age-matched controls (housed under conventional 12 hour/day illumination), and urothelial Panx1 mRNA levels were quantified by real time PCR. Cystometric data from an additional group of WT mice was obtained pre-CI and then post-CI with and without intravesical treatment with Panx1 channel blocker mefloquine (300nM). Data is expressed as mean±SEM and statistical difference determined by paired or unpaired Student’s t-test. RESULTS: In WT mice, pelvic tactile threshold decreased 18-fold (pre-CI: 0.22 ± 0.06 vs post-CI: 0.012 ± 0.007; N = 4 p<0.01) and voiding frequency increased 2-fold (pre-CI: 0.56 ± 0.04 vs post-CI: 1.11 ± 0.12; N = 4 p<0.01) after CI. This was accompanied by a 2.5-fold increase in Panx1 expression in the bladder urothelium when compared to non-stressed WT mice (2.53 ± 0.29 stressed/non-stressed controls; N=4; p<0.001), and a 2-fold increase in urothelial ATP release compared to baseline levels (1.8 ± 0.24 post-CI/pre-CI; N = 4; p=0.007). Notably, emergence of pelvic pain and voiding frequency was abrogated in Panx1-null mice submitted to CI stress, and urothelial ATP release was not different from control pre-CI levels. Urodynamic assessment of WT mice after CI indicated marked bladder hyperactivity that was normalized by intravesical treatment with Panx1 channel blocker mefloquine. CONCLUSIONS: Panx1 channels play a significant role in stress-induced pelvic pain and urinary frequency, likely by amplifying urothelial ATP release that can ultimately change the bladder sensory and motor responses, and cause bladder sensitization. Source of Funding: None

Mechanism of Action of Botulinum Toxin A in Treatment of Functional Urological Disorders

Intravesical botulinum toxin (BoNT) injection is effective in reducing urgency and urinary incontinence. It temporarily inhibits the detrusor muscle contraction by blocking the release of acetylcholine (Ach) from the preganglionic and postganglionic nerves in the efferent nerves. BoNT-A also blocks ATP release from purinergic efferent nerves in the detrusor muscle. In afferent nerves, BoNT-A injection markedly reduces the urothelial ATP release and increases nitric oxide (NO) release from the urothelium. BoNT-A injection in the urethra or bladder has been developed in the past few decades as the treatment method for detrusor sphincter dyssyndergia, incontinence due to neurogenic or idiopathic detrusor overactivity, sensory disorders, including bladder hypersensitivity, overactive bladder, and interstitial cystitis/chronic pelvic pain syndrome. Although the FDA only approved BoNT-A injection treatment for neurogenic detrusor overactivity and for refractory overactive bladder, emerging clinical trials have demonstrated the benefits of BoNT-A treatment in functional urological disorders. Cautious selection of patients and urodynamic evaluation for confirmation of diagnosis are crucial to maximize the successful outcomes of BoNT-A treatment.

Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice.

Background and purposePDE inhibitors such as sildenafil alleviate lower urinary tract symptoms; however, a complete understanding of their action on the bladder remains unclear. We are investigating the effects of sildenafil, on post and preganglionic nerve‐mediated contractions of the mouse bladder, and neuronal and urothelial ATP release.Experimental approachBladders were used from young (12 weeks), aged (24 months), and spinal cord transected (SCT), mice, for in vitro contractility experiments. An arterially perfused in situ whole mouse model was used to record bladder pressure. Nerve‐mediated contractions were generated by electrical field stimulation (EFS) of postganglionic nerve terminals or the pelvic nerve. ATP release during EFS in intact detrusor strips, and during stretch of isolated mucosa strips, was measured using a luciferin‐luciferase assay.Key resultsSildenafil (20 μM) inhibited nerve‐mediated contractions in young mice, with an increase in f 1/2 values in force–frequency relationships, demonstrating a greater effect at low frequencies. Sildenafil reduced the atropine‐resistant, purinergic component of nerve‐mediated contractions, and suppressed neuronal ATP release upon EFS in vitro. Sildenafil reduced the preganglionic pelvic nerve stimulated bladder pressure recordings in situ; comparable to in vitro experiments. Sildenafil reduced stretch‐induced urothelial ATP release. Sildenafil also relaxed nerve‐mediated contractions in aged and SCT mice.Conclusion and implicationsSildenafil has a greater effect on the low‐frequency, purinergic‐mediated contractions and suppresses neuronal ATP release. In addition, sildenafil reduces stretch‐induced urothelial ATP release. These results demonstrate a novel action of sildenafil to selectively inhibit ATP release from nerve terminals innervating detrusor smooth muscle and the urothelium.

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.

Gemcitabine: Selective cytotoxicity, induction of inflammation and effects on urothelial function

Intravesical gemcitabine has recently been introduced for the treatment of superficial bladder cancer and has a favourable efficacy and toxicity profile in comparison to mitomycin c (MMC), the most commonly used chemotherapeutic agent. The aim of this study was to assess the cytotoxic potency of gemcitabine in comparison to MMC in urothelial cell lines derived from non-malignant (UROtsa) and malignant (RT4 and T24) tissues to assess selectivity. Cells were treated with gemcitabine or mitomycin C at concentrations up to the clinical doses for 1 or 2h respectively (clinical duration). Treatment combined with hyperthermia was also examined. Cell viability, ROS formation, urothelial function (ATP, acetylcholine and PGE2 release) and secretion of inflammatory cytokines were assessed. Gemcitabine displayed a high cytotoxic selectivity for the two malignant cell lines (RT4, T24) compared to the non-malignant urothelial cells (UROtsa, proliferative and non-proliferative). In contrast, the cytotoxic effects of MMC were non-selective with equivalent potency in each of the cell lines. The cytotoxic effect of gemcitabine in the malignant cell lines was associated with an elevation in free radical formation and was significantly decreased in the presence of an equilibrative nucleoside transporter inhibitor. Transient changes in urothelial ATP and PGE2 release were observed, with significant increase in release of interleukin-6, interleukin-8 and interleukin-1β from urothelial cells treated with gemcitabine. The selectivity of gemcitabine for malignant urothelial cells may account for the less frequent adverse urological effects with comparison to other commonly used chemotherapeutic agents.

Purinergic signalling underlies transforming growth factor-β-mediated bladder afferent nerve hyperexcitability.

The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor-β1 (TGF-β1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP-induced cystitis is decreased with TGF-β inhibition. These results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling. The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor-β (TGF-β) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF-β in bladder inflammation, the precise mechanisms of TGF-β mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation. We utilized bladder-pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF-β1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signalling in cyclophosphamide-induced cystitis with TβR-1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF-β-mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.

Serotonergic regulation of distention-induced ATP release from the urothelium.

Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT1D and 5-HT4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH2O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT1B/1D antagonist GR-127935 but not by the 5-HT4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT1D.

Polarized ATP distribution in urothelial mucosal and serosal space is differentially regulated by stretch and ectonucleotidases.

Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.

Purinergic signalling in the urinary bladder

It is well established that in most species, exocytotic vesicular release of ATP from parasympathetic neurons contributes to contraction of the bladder. However, ATP is released not only from parasympathetic nerves, but also from the urothelium. During bladder filling, the urothelium is stretched and ATP is released from the umbrella cells thereby activating mechanotransduction pathways. ATP release can also be induced by various mediators present in the urine and and/or released from nerves or other components of the lamina propria. Urothelial release of ATP is mainly attributable to vesicular transport or exocytosis and, to a smaller extent, to pannexin hemichannel conductive efflux. After release, ATP acts on P2X3 and P2X2/3 receptors on suburothelial sensory nerves to initiate the voiding reflex and to mediate the sensation of bladder filling and urgency. ATP also acts on suburothelial interstitial cells/myofibroblasts generating an inward Ca(2+) transient that via gap junctions could provide a mechanism for long-distance spread of signals from the urothelium to the detrusor muscle. ATP release can be affected by urological diseases, e.g., interstitial cystitis and both the mechanisms of release and the receptors activated by ATP may be targets for future drugs for treatment of lower urinary tract disorders.

MP12-19 SPONTANEOUS MICTURITION BEHAVIOR AND CYSTOMETRIC EVALUATION OF BLADDER FUNCTION IN P2X7 RECEPTOR DEFICIENT MICE

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research II1 Apr 2015MP12-19 SPONTANEOUS MICTURITION BEHAVIOR AND CYSTOMETRIC EVALUATION OF BLADDER FUNCTION IN P2X7 RECEPTOR DEFICIENT MICE Hiromitsu Negoro, Nuan Cui, Osamu Ogawa, and Sylvia Suadicani Hiromitsu NegoroHiromitsu Negoro More articles by this author , Nuan CuiNuan Cui More articles by this author , Osamu OgawaOsamu Ogawa More articles by this author , and Sylvia SuadicaniSylvia Suadicani More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.794AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES ATP signaling plays major roles in the bladder sensory and motor functions. Several P2 receptor (P2R) subtypes are expressed in the bladder but little is till known of their actual individual role in bladder function and dysfunction. Studies have indicated a role for P2X7R in hemorrhagic cystitis, and recently the P2X7R participation in urothelial mechanosensory transduction. We have investigated the extent to which deletion of P2X7R affects bladder function. METHODS Three months old female wild-type and P2X7R-null mice were used. Expression of P2X7R was assessed by immunoblotting. Spontaneous micturition behavior was assessed using the automated voided stain on paper (aVSOP) method. Cystometric parameters measured: Bladder capacity (BC, µL), micturition volume (MV, µL), residual volume (RV; µL), bladder compliance (BCo, μL/cmH2O), basal pressure (BP; cmH2O), threshold pressure (TP; cmH2O) and micturition pressure (MP; cmH2O). Effects of P2X7R deletion on bladder function was also evaluated as changes in intravesical stretch-induced ATP release. RESULTS P2X7R expression in mucosa and detrusor was confirmed in WT and no expression in P2X7R-null mice. Body weight and gross bladder morphology did not differ between WT and P2X7R-null. ATP release was 60% lower in P2X7R-null compared to WT (WT: 0.9±0.2nM vs P2X7R: 0.3±0.03nM; N=9 and 7, P<0.05). aVSOP analysis demonstrated that voided volume per micturition was significantly higher in P2X7R-null than in WT but frequency and total urine volume were not (average of 4 hours, N=3 and 5, P<0.05, shown in figure). Cystometric analysis indicated that P2X7R-null mice display higher BC (WT: 127.1±4.7; P2X7R: 187.9±3.3; N=6 and 6, P<0.0001), MV (WT: 81.0±4.6; P2X7R: 142.0±4.3, P<0.0001), MP (WT: 25.4±0.6, P2X7R: 31.3±0.8, P<0.0001), TP (WT: 18.3±0.5, P2X7R: 21.5±0.8; P<0.01) and BCo (WT: 16.3±2.2; P2X7R: 70.7±12.7, P<0.001). BP was lower in P2X7R-null compared to WT mice (WT: 7.9±0.2; P2X7R: 6.0±0.2, P<0.0001). RV was similar (WT: 45.9±3.2; P2X7R: 44.1±3.5). CONCLUSIONS P2X7R deficient mice display significant changes in bladder function. These changes likely result from decreased urothelial ATP release and signaling, and consequent reduced sensory activation in response to bladder distension. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e135 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiromitsu Negoro More articles by this author Nuan Cui More articles by this author Osamu Ogawa More articles by this author Sylvia Suadicani More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1087 A novel mechanism of infection-induced bladder hyperactivity: LPS causes rapid urothelial ATP release and stimulates the mechanosensory afferent pathway

1087 A novel mechanism of infection-induced bladder hyperactivity: LPS causes rapid urothelial ATP release and stimulates the mechanosensory afferent pathway