Blockade of Acid-Sensing Ion Channels Increases Urinary Bladder Capacity With or Without Intravesical Irritation in Mice.

We conducted this study to examine whether acid-sensing ion channels (ASICs) are involved in the modulation of urinary bladder activity with or without intravesical irritation induced by acetic acid. All in vivo evaluations were conducted during continuous infusion cystometry in decerebrated unanesthetized female mice. During cystometry with a pH 6.3 saline infusion, an i.p. injection of 30 µmol/kg A-317567 (a potent, non-amiloride ASIC blocker) increased the intercontraction interval (ICI) by 30% (P < 0.001), whereas vehicle injection had no effect. An intravesical acetic acid (pH 3.0) infusion induced bladder hyperactivity, with reductions in ICI and maximal voiding pressure (MVP) by 79% (P < 0.0001) and 29% (P < 0.001), respectively. A-317567 (30 µmol/kg i.p.) alleviated hyperreflexia by increasing the acid-shortened ICI by 76% (P < 0.001). This dose produced no effect on MVP under either intravesical pH condition. Further analysis in comparison with vehicle showed that the increase in ICI (or bladder capacity) by the drug was not dependent on bladder compliance. Meanwhile, intravesical perfusion of A-317567 (100 µM) had no effect on bladder activity during pH 6.0 saline infusion cystometry, and drug perfusion at neither 100 µM nor 1 mM produced any effects on bladder hyperreflexia during pH 3.0 acetic acid infusion cystometry. A-317567 has been suggested to display extremely poor penetrability into the central nervous system and thus to be a peripherally active blocker. Taken together, our results suggest that blockade of ASIC signal transduction increases bladder capacity under normal intravesical pH conditions and alleviates bladder hyperreflexia induced by intravesical acidification and that the site responsible for this action is likely to be the dorsal root ganglia.

Best practices for cystometric evaluation of lower urinary tract function in muriform rodents.

AIMS: Rodent cystometry has provided valuable insights into the impact of the disease, injury, and aging on the cellular and molecular pathways, neurologic processes, and biomechanics of lower urinary tract function. The purpose of this white paper is to highlight the benefits and shortcomings of different experimental methods and strategies and to provide guidance on the proper interpretation of results. METHODS: Literature search, selection of articles, and conclusions based on discussions among a panel of workers in the field. RESULTS: A range of cystometric tests and techniques used to explore biological phenomena relevant to the lower urinary tract are described, the advantages and disadvantages of various experimental conditions are discussed, and guidance on the practical aspects of experimental execution and proper interpretation of results are provided. CONCLUSIONS: Cystometric evaluation of rodents comprises an extensive collection of functional tests that can be performed under a variety of experimental conditions. Decisions regarding which approaches to choose should be determined by the specific questions to be addressed and implementation of the test should follow standardized procedures.

Corrigendum: Blockade of Acid-Sensing Ion Channels Increases Urinary Bladder Capacity With or Without Intravesical Irritation in Mice.

[This corrects the article DOI: 10.3389/fphys.2020.592867.].

The effect of Ferula elaeochytris root extract on erectile dysfunction in streptozotocin-induced diabetic rat.

Erectile dysfunction (ED) is an important complication of diabetes. The aim of our study was to determine whether Ferula elaeochytris (FE) root extract could affect ED in streptozotocin (STZ)-induced diabetic rats. Seventy-five adult male Wistar albino rats were equally divided into five groups; control (C), FE (40 mg/kg-d), STZ-induced diabetes (60 mg/kg) (DM), diabetes + F. elaeochytris (DM + FE), and ethanol (EtOH). After 8 weeks, in vitro and in vivo parameters (intracavernosal pressure [ICP]), testicle and body weight, serum glucose levels, and histopathology were assessed. In the STZ-induced diabetic group, acetylcholine-induced endothelium-dependent relaxation responses, and electrical field stimulation-induced neurogenic and nitrergic relaxation responses were decreased significantly, while FE administration to diabetic rats reversed the decreased nitrergic and neurogenic responses. In the diabetic group, ICP/MAP (0.1375 ± 0.02 cm/H2O), spermatogenesis in testicles (53.73 ± 0.81), and testicle weights (257.8 ± 20.63) were decreased significantly; however, FE administration to diabetic rats restored the decreased values (0.350 ± 0.019 cm/H2O, 75.07 ± 0.35, and 416 ± 24.11, respectively). In the DM group, blood glucose levels were increased (411.7 ± 18.30) compared to the C group. However, FE administration to diabetic rats reduced glucose levels (230.6 ± 25.60 mg/dL) compared to the DM group. In conclusion, FE recovered neurogenic and endothelial dysfunction and decreased glucose levels in diabetic rats.

Author Correction: Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia.

Intermittent stress disrupts the circadian rhythm in clock genes such as Per2 only in peripheral organs without any effect on the central circadian clock in the suprachiasmatic nucleus. Here, the effect of restraint stress (RS) on circadian bladder function was investigated based on urination behavior and gene expression rhythms. Furthermore, PF670462 (PF), a Per2 phosphorylation enzyme inhibitor, was administered to investigate the effects on circadian bladder re-alignment after RS. Two-hour RS during the light (sleep) phase was applied to mice (RS mice) for 5 days. The following parameters were then examined: urination behaviors; clock gene expression rhythms and urinary sensory-related molecules such as piezo type mechanosensitive ion channel component 1 (Piezo1), transient receptor potential cation channel subfamily V member 4 (TRPV4), and Connexin26 (Cx26) in the bladder mucosa; Per2 expression in the excised bladder of Per2luciferase knock-in mice (Per2::luc); in vivo Per2 expression rhythms in the bladder of Per2::luc mice. Control mice did not show altered urination behavior in the light phase, whereas RS mice exhibited a higher voiding frequency and lower bladder capacity. In the bladder mucosa, RS mice also showed abrogated or misaligned Piezo1, TRPV4, Connexin26, and clock gene expression. The rhythmic expression of Per2 was also altered in RS mice both in excised- and in vivo bladder, compared with control mice. After PF administration, voiding frequency was reduced and bladder capacity was increased during the light phase in RS mice; the in vivo Per2 expression rhythm was also fully restored. Therefore, RS can alter circadian gene expression in the bladder during the light phase and might cause nocturia via changes in circadian bladder function due the dysregulation of clock genes. Amending the circadian rhythm therapeutically could be applied for nocturia.

The time-dependent variation of ATP release in mouse primary-cultured urothelial cells is regulated by the clock gene.

AIMS: The sensation of bladder fullness (SBF) is triggered by the release of ATP. Therefore, the aim of this study was to investigate whether time-dependent changes in the levels of stretch-released ATP in mouse primary-cultured urothelial cells (MPCUCs) is regulated by circadian rhythm via clock genes. METHODS: MPCUCs were derived from wild-type and Clock mutant mice (ClockΔ19/Δ19 ), presenting a nocturia phenotype. They were cultured in elastic silicone chambers. Stretch-released ATP was quantified every 4 h by ATP photon count. An experiment was also performed to determine whether ATP release correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Connexin26 (Cx26) in MPCUCs regulated by clock genes with circadian rhythms. MPCUCs were treated with carbenoxolone, an inhibitor of gap junction protein; were derived from VNUT-KO mice; or treated with Piezo1-siRNA, TRPV4-siRNA, and Cx26-siRNA. RESULTS: Stretch-released ATP showed time-dependent changes in wild-type mice and correlated with the rhythm of the expression of Piezo1, TRPV4, VNUT, and Cx26. However, these rhythms were disrupted in ClockΔ19/Δ19 mice. Carbenoxolone eliminated the rhythmicity of ATP release in wild-type mice. However, time-dependent ATP release changes were maintained when a single gene was deficient such as VNUT-KO, Piezo1-, TRPV4-, and Cx26-siRNA. CONCLUSIONS: ATP release in the bladder urothelium induces SBF and may have a circadian rhythm regulated by the clock genes. In the bladder urothelium, clock gene abnormalities may disrupt circadian ATP release by inducing Piezo1, TRPV4, VNUT, and Cx26. All these genes can trigger nocturia.

The oscillation of intracellular Ca2+ influx associated with the circadian expression of Piezo1 and TRPV4 in the bladder urothelium.

We previously showed that bladder functions are controlled by clock genes with circadian rhythm. The sensation of bladder fullness (SBF) is sensed by mechano-sensor such as Piezo1 and TRPV4 in the mouse bladder urothelium. However, functional circadian rhythms of such mechano-sensors remain unknown. To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+] i ) using mouse primary cultured urothelial cells (MPCUCs). Using Ca2+ imaging, stretch-evoked [Ca2+] i was quantified every 4 h in MPCUCs derived from wild-type (WT) and Clock Δ19/Δ19 mice, which showed a nocturia phenotype. Furthermore, a Piezo1 inhibitor GsMTx4 and a TRPV4 inhibitor Ruthenium Red were applied and stretch-evoked [Ca2+] i in MPCUCs was measured to investigate their contribution to SBF. Stretch-evoked [Ca2+] i showed a circadian rhythm in the WT mice. In contrast, Clock Δ19/Δ19 mice showed disrupted circadian rhythm. The administration of both GsMTx4 and Ruthenium Red eliminated the circadian rhythm of stretch-evoked [Ca2+] i in WT mice. We conclude that SBF may have a circadian rhythm, which is created by functional circadian changes of Piezo1 and TRPV4 being controlled by clock genes to be active during wakefulness and inactive during sleep. Abnormalities of clock genes disrupt SBF, and induce nocturia.

Role of proNGF/p75 signaling in bladder dysfunction after spinal cord injury.

Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro-nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase-positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.

The Circadian expression of Piezo1, TRPV4, Connexin26, and VNUT, associated with the expression levels of the clock genes in mouse primary cultured urothelial cells.

AIMS: To investigate circadian gene expressions in the mouse bladder urothelium to establish an experimental model and study the functions of the circadian rhythm. METHODS: The gene expression rhythms of the clock genes, mechano-sensors such as Piezo1 and TRPV4, ATP release mediated molecules (ARMM) such as Cx26 and VNUT were investigated in mouse primary cultured urothelial cells (UCs) of wild-type (WT) and Clock mutant (ClockΔ19/Δ19 ) mice using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting analysis. The long-term oscillation of the clock genes in UC was investigated by measuring bioluminescence from UC isolated from Period2luciferase knock-in mice (Per2::luc) and Per2::luc with ClockΔ19/Δ19 using a luminometer. The mRNA expression rhythms after treatment with Clock short interfering RNA (siRNA) were also measured to compare differences between Clock point mutations and Clock deficiency. RESULTS: The UCs from WT mice showed the time-dependent gene expressions for clock genes, mechano-sensors, and ARMM. The abundances of the products of these genes also correlated with the mRNA expression rhythms in UCs. The bioluminescence of Per2::Luc in UCs showed a circadian rhythm. By contrast, all the gene expressions rhythms observed in WT mice were abrogated in the ClockΔ19/Δ19 mice. Transfection with Clock siRNA in UCs had the same effect as the Clock mutation. CONCLUSIONS: We demonstrated that the time-dependent gene expressions, including clock genes, mechano-sensors, and ARMM, were reproducible in UCs. These findings demonstrated that UCs have the potential to progress research into the circadian functions of the lower urinary tract regulated by clock genes.

The role of capsaicin-sensitive C-fiber afferent pathways in the control of micturition in spinal-intact and spinal cord-injured mice.

We examined bladder and urethral sphincter activity in mice with or without spinal cord injury (SCI) after C-fiber afferent desensitization induced by capsaicin pretreatment and changes in electrophysiological properties of mouse bladder afferent neurons 4 wk after SCI. Female C57BL/6N mice were divided into four groups: 1) spinal intact (SI)-control, 2) SI-capsaicin pretreatment (Cap), 3) SCI-control, and 4) SCI-Cap groups. Continuous cystometry and external urethral sphincter (EUS)-electromyogram (EMG) were conducted under an awake condition. In the Cap groups, capsaicin (25, 50, or 100 mg/kg) was injected subcutaneously 4 days before the experiments. In the SI-Cap group, 100 mg/kg capsaicin pretreatment significantly increased bladder capacity and decreased the silent period duration of EUS/EMG compared with the SI-control group. In the SCI-Cap group, 50 and 100 mg/kg capsaicin pretreatment decreased the number of nonvoiding contractions (NVCs) and the duration of reduced EUS activity during voiding, respectively, compared with the SCI-control group. In SCI mice, hexamethonium, a ganglionic blocker, almost completely blocked NVCs, suggesting that they are of neurogenic origin. Patch-clamp recordings in capsaicin-sensitive bladder afferent neurons from SCI mice showed hyperexcitability, which was evidenced by decreased spike thresholds and increased firing rate compared with SI mice. These results indicate that capsaicin-sensitive C-fiber afferent pathways, which become hyperexcitable after SCI, can modulate bladder and urethral sphincter activity in awake SI and SCI mice. Detrusor overactivity as shown by NVCs in SCI mice is significantly but partially dependent on capsaicin-sensitive C-fiber afferents, whereas the EUS relaxation during voiding is enhanced by capsaicin-sensitive C-fiber bladder afferents in SI and SCI mice.

Ketanserin and Naftopidil Enhance the Potentiating Effect of Alpha-Methyl-Serotonin on the Neurally-Induced Contraction of Human Isolated Urinary Bladder Muscle Strips.

PURPOSE: The aim of this study was to assess the potential involvement of a specific subtype of 5-hydroxytryptamine (5-HT), 5HT2 receptors in neurally-induced contractions of the human detrusor. METHODS: Contractile responses to electrical field stimulation (EFS) were examined in human isolated urinary bladder muscle strips. The potentiation of EFS-induced detrusor contraction was examined by adding cumulative concentrations of a 5-HT and 5-HT2 receptor agonist, α-methyl-serotonin (α-Me-5-HT) (1nM-100µM) in the presence or absence of a 5-HT2 antagonist, ketanserin (5-HT2A>5-HT2C) or naftopidil (5-HT2B>5-HT2A) (0.3-3µM). RESULTS: 5-HT and α-Me-5-HT potentiated EFS-induced contraction with a maximal effect (Emax) of 37.6% and 38.6%, respectively, and with pEC50 (negative logarithm of the concentration required for a half-maximal response to an agonist) values of 8.3 and 6.8, respectively. Neither ketanserin nor naftopidil at any concentration produced a rightward displacement of the α-Me-5-HT concentration response curve. Instead, the Emax of α-Me-5-HT increased in the presence of ketanserin at 0.3-1µM and in the presence of naftopidil at 1µM to 51% and 56%, respectively, while the Emax in the presence of vehicle alone was 36%. The highest concentration (3µM) of either drug, however, fully reversed the enhancement. CONCLUSIONS: The potentiating effect of α-Me-5-HT on neurally-induced contraction of human urinary bladder muscle strips was not found to be mediated via any 5-HT2 receptor subtypes. The underlying mechanism for the enhancement of the α-Me-5-HT potentiating effect on detrusor contractility by ketanserin and naftopidil remains unknown; however, our results suggest that these drugs may be useful for treating contractile dysfunction of the detrusor, as manifested in conditions such as underactive bladder.

P2Y6-deficiency increases micturition frequency and attenuates sustained contractility of the urinary bladder in mice.

The role of the P2Y6 receptor in bladder function has recently attracted a great deal of attention in lower urinary tract research. We conducted this study to determine contributions of the P2Y6 receptor in lower urinary tract function of normal phenotypes by comparing P2Y6-deficient mice and wild-type mice. In in vivo experiments, P2Y6-deficient mice had more frequent micturition with smaller bladder capacity compared to wild-type mice; however, there was no difference between these groups in bladder-filling pressure/volume relationships during cystometry under decerebrate, unanaesthetized conditions. Analysis of in vivo bladder contraction revealed significant difference between the 2 groups, with P2Y6-deficient mice presenting markedly shorter bladder contraction duration but no difference in peak contraction pressure. However, analysis of in vitro experiments showed no P2Y6 involvements in contraction and relaxation of bladder muscle strips and in ATP release by mechanical stimulation of primary-cultured urothelial cells. These results suggest that the P2Y6 receptor in the central nervous system, dorsal root ganglion, or both is involved in inhibition of bladder afferent signalling or sensitivity in the pontine micturition centre and that the receptor in the detrusor may be implicated in facilitation to sustain bladder contraction force.

Clock Genes Regulate the Circadian Expression of Piezo1, TRPV4, Connexin26, and VNUT in an Ex Vivo Mouse Bladder Mucosa.

OBJECTIVES: ClockΔ19/Δ19 mice is an experimental model mouse for nocturia (NOC). Using the bladder mucosa obtained from ClockΔ19/Δ19 mice, we investigated the gene expression rhythms of mechanosensory cation channels such as transient receptor potential cation channel subfamily V member 4 (TRPV4) and Piezo1, and main ATP release pathways including vesicular nucleotide transporter (VNUT) and Connexin26(Cx26), in addition to clock genes. MATERIALS AND METHODS: Eight- to twelve-week-old male C57BL/6 mice (WT) and age- and sex-matched C57BL/6 ClockΔ19/Δ19 mice, which were bred under 12-h light/dark conditions for 2 weeks, were used. Gene expression rhythms and transcriptional regulation mechanisms in clock genes, mechanosensor, Cx26 and VNUT were measured in the mouse bladder mucosa, collected every 4 hours from WT and ClockΔ19/Δ19 mice using quantitative RT-PCR, a Western blot analysis, and ChIP assays. RESULTS: WT mice showed circadian rhythms in clock genes as well as mechanosensor, Cx26 and VNUT. Their expression was low during the sleep phase. The results of ChIP assays showed Clock protein binding to the promotor regions and the transcriptional regulation of mechanosensor, Cx26 and VNUT. In contrast, all of these circadian expressions were disrupted in ClockΔ19/Δ19 mice. The gene expression of mechanosensor, Cx26 and VNUT was maintained at a higher level in spite of the sleep phase. CONCLUSIONS: Mechanosensor, Cx26 and VNUT expressed with circadian rhythm in the mouse bladder mucosa. The disruption of circadian rhythms in these genes, induced by the abnormalities in clock genes, may be factors contributing to NOC because of hypersensitivity to bladder wall extension.

The Clock mutant mouse is a novel experimental model for nocturia and nocturnal polyuria.

AIMS: The pathophysiologies of nocturia (NOC) and nocturnal polyuria (NP) are multifactorial and their etiologies remain unclear in a large number of patients. Clock genes exist in most cells and organs, and the products of Clock regulate circadian rhythms as representative clock genes. Clock genes regulate lower urinary tract function, and a newly suggested concept is that abnormalities in clock genes cause lower urinary tract symptoms. In the present study, we investigated the voiding behavior of Clock mutant (ClockΔ19/Δ19 ) mice in order to determine the effects of clock genes on NOC/NP. METHODS: Male C57BL/6 mice aged 8-12 weeks (WT) and male C57BL/6 ClockΔ19/Δ19 mice aged 8 weeks were used. They were bred under 12 hr light/dark conditions for 2 weeks and voiding behavior was investigated by measuring water intake volume, urine volume, urine volume/void, and voiding frequency in metabolic cages in the dark and light periods. RESULTS: No significant differences were observed in behavior patterns between ClockΔ19/Δ19 and WT mice. ClockΔ19/Δ19 mice showed greater voiding frequencies and urine volumes during the sleep phase than WT mice. The diurnal change in urine volume/void between the dark and light periods in WT mice was absent in ClockΔ19/Δ19 mice. Additionally, functional bladder capacity was significantly lower in ClockΔ19/Δ19 mice than in WT mice. CONCLUSIONS: We demonstrated that ClockΔ19/Δ19 mice showed the phenotype of NOC/NP. The ClockΔ19/Δ19 mouse may be used as an animal model of NOC and NP. Neurourol. Urodynam. 36:1034-1038, 2017. © 2016 Wiley Periodicals, Inc.

Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs.

Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA). Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of AA. After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v.) or L-733,060 (3-10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit and therefore may be useful clinically in treating bladder overactivity symptoms.

Urothelial ATP exocytosis: regulation of bladder compliance in the urine storage phase.

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.

Characterization of bladder and external urethral activity in mice with or without spinal cord injury--a comparison study with rats.

To clarify the lower urinary tract function in mice, we compared bladder and urethral activity between rats and mice with or without spinal cord injury (SCI). Female Sprague-Dawley rats and C57BL/6N mice were divided into five groups:1) spinal intact (SI) rats,2) SI mice,3) pudendal nerve transection (PNT) SI mice,4) spinal cord injury (SCI) rats, and 5) SCI mice. Continuous cystometry (CMG) and external urethral sphincter (EUS)-electromyogram (EMG) analyses were conducted under an awake, restrained condition. During voiding bladder contractions, SI animals exhibited EUS bursting with alternating active and silent periods, which, in rats but not mice, coincided with small-amplitude intravesical pressure oscillations in CMG recordings. In SI mice with bursting-like EUS activity, the duration of active periods was significantly shorter by 46% (32 ± 5 ms) compared with SI rats (59 ± 9 ms). In PNT-SI mice, there were no significant differences in any of cystometric parameters compared with SI mice. In SCI rats, fluid elimination from the urethra and the EUS bursting occurred during small-amplitude intravesical pressure oscillations. However, SCI mice did not exhibit clear EUS bursting activity or intravesical pressure oscillations but rather exhibited intermittent voiding with slow large-amplitude reductions in intravesical pressure, which occurred during periods of reduced EUS activity. These results indicate that EUS pumping activity is essential for generating efficient voiding in rats with or without spinal cord injury. However, EUS bursting activity is not required for efficient voiding in SI mice and does not reemerge in SCI mice in which inefficient voiding occurs during periods of reduced tonic EUS activity.

Functional roles of TRPV1 and TRPV4 in control of lower urinary tract activity: dual analysis of behavior and reflex during the micturition cycle.

The present study used a dual analysis of voiding behavior and reflex micturition to examine lower urinary tract function in transient receptor potential vanilloid (TRPV)1 knockout (KO) mice and TRPV4 KO mice. In metabolic cage experiments conducted under conscious conditions (i.e., voluntary voiding behavior), TRPV4 KO mice showed a markedly higher voiding frequency (VF; 19.3 ± 1.2 times/day) and a smaller urine volume/voiding (UVV; 114 ± 9 µl) compared with wild-type (WT) littermates (VF: 5.2 ± 0.5 times/day and UVV: 380 ± 34 µl). Meanwhile, TRPV1 KO mice showed a similar VF to WT littermates (6.8 ± 0.5 times/day) with a significantly smaller UVV (276 ± 20 µl). Water intake among these genotypes was the same, but TRPV4 KO mice had a larger urine output than the other two groups. In cystometrogram experiments conducted in decerebrate unanesthetized mice (i.e., reflex micturition response), no differences between the three groups were found in any cystometrogram variables, including voided volume, volume threshold for inducing micturition contraction, maximal voiding pressure, and bladder compliance. However, both TRPV1 KO and TRPV4 KO mice showed a significant number of nonvoiding bladder contractions (NVCs; 3.5 ± 0.9 and 2.8 ± 0.7 contractions, respectively) before each voiding, whereas WT mice showed virtually no NVCs. These results suggest that in the reflex micturition circuit, a lack of either channel is involved in NVCs during bladder filling, whereas in the forebrain, it is involved in the early timing of urine release, possibly in the conscious response to the bladder instability.

Effects of urethane on reflex activity of lower urinary tract in decerebrate unanesthetized rats.

Effects of urethane on lower urinary tract function were examined in decerebrate unanesthetized rats. During single slow infusion (0.04 ml/min) cystometrograms (CMGs), urethane (0.3 g/kg) increased micturition pressure threshold (PT) by 73%, postvoid residual volume (RV) by 425%, and decreased voiding efficiency (VE) by 57%, but did not change maximal voiding pressure (MVP), closing peak pressure (CPP), bladder compliance, bladder contraction duration (BCD), or volume threshold (VT) for inducing micturition. Lower doses (0.01-0.1 g/kg) did not alter any parameter. During continuous fast infusion (0.21 ml/min) CMGs, urethane at doses of 0.6-1.2 g/kg (iv) markedly decreased CPP by 69-85%, whereas only the largest dose (1.2 g/kg iv) decreased MVP and external urethral sphincter electromyogram activity by 42 and by 80%, respectively. Doses of 0.001-0.6 g/kg did not alter the intercontraction interval and BCD. Taken together, these results suggest that urethral activity, which is essential for efficient voiding, is more sensitive to the suppressive effect of urethane than afferent or efferent mechanisms controlling the bladder. The threshold dose of MK-801 (0.3 mg/kg), an NMDA antagonist, required to decrease MVP and increase VT in urethane (1.2 g/kg)-anesthetized rats, only increased VT in rats treated with a subanesthetic dose of urethane (0.3 g/kg), suggesting a higher sensitivity of the afferent vs. efferent limb of the micturition reflex pathway to urethane-MK-801 interactions. Because effects of urethane persisted after removal of the forebrain, they must be mediated by actions on the brain stem, spinal cord, or peripheral nervous system.