Urinary Bladder Contractility Research Articles

Compound 48/80 causes overactive bladder without altering urothelial permeability in mice

The urothelium, a transitional epithelium lining the urinary bladder, is essential for preventing leakage of urine into the underlying tissue. Disruption of this barrier by noxious stimuli or inflammatory mediators increases bladder wall permeability and causes bladder dysfunction. Compound 48/80 is a pro-inflammatory basic secretagogue that causes urothelium-dependent increases in urinary bladder contractility, as well as bladder overactivity in mice. We hypothesized that Compound 48/80 causes bladder dysfunction by increasing urothelial permeability. Intact bladders from male C57BL/6 mice (N=4) were cannulated, filled with HEPES-buffered physiological salt solution containing FITC-labeled-dextran (70 kDa), and pressurized to 25 mmHg. Z-stacks of baseline fluorescence were captured across the bladder wall using 2-photon laser microscopy. Compound 48/80 (50 μg/mL) was then instilled intravesically for 10 minutes before the bladder wall was imaged again. Average FITC intensity plots wall showed no consistent increase in fluorescence with or without Compound 48/80 from the lumen into the bladder wall. There was also no statistical difference in the area under the curve of these line graphs (p=0.5898). Our findings suggest that Compound 48/80 causes overactive bladder without affecting urothelial permeability. Future studies will investigate other secretagogues and lower molecular weight dextrans to clarify the mechanisms by which secretagogues cause a loss of urothelial barrier function. This research is supported by NIH P20-DK127554 (NRT), NIH R01-DK119615 (NRT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.

Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4-chloro-2-[2-(2-chloro-phenoxy)-acetylamino]-benzoic acid (CBA) versus classical 9-phenanthrol.

Non‐selective cation channels in urinary bladder smooth muscle (UBSM) are thought to mediate increases in cellular excitability and contractility. For transient receptor potential melastatin type‐4 (TRPM4) channels, the evidence primarily relies on the inhibitor 9‐phenanthrol, which exhibits pharmacological limitations. Recently, 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) has been discovered as a novel TRPM4 channel blocker. We examined how, in comparison to 9‐phenanthrol, CBA affects the excitability of freshly isolated guinea pig UBSM cells and the contractility of UBSM strips. Additionally, non‐selective TRPM4 channel inhibitor flufenamic acid (FFA) and potentiator BTP2 (also known as YM‐58483) were studied in UBSM cells. Unlike robust inhibition for 9‐phenanthrol already known, CBA (up to 100 μM) displayed either no or a very weak reduction (<20%) in spontaneous phasic, 20 mM KCl‐induced, and electrical field stimulated contractions. For 300 μM CBA, reductions were higher except for an increase in the frequency of KCl‐induced contractions. In UBSM cells, examined under amphotericin B‐perforated patch‐clamp, CBA (30 μM) did not affect the membrane potential (I = 0) or voltage step‐induced whole‐cell cation currents, sensitive to 9‐phenanthrol. The currents were not inhibited by FFA (100 μM), increased by BTP2 (10 μM), nor enhanced under a strongly depolarizing holding voltage of −16 or + 6 mV (vs. −74 mV). None of the three compounds affected the cell capacitance, unlike 9‐phenanthrol. In summary, the novel inhibitor CBA and nonselective FFA did not mimic the inhibitory properties of 9‐phenanthrol on UBSM function. These results suggest that TRPM4 channels, although expressed in UBSM, play a distinct role rather than direct regulation of excitability and contractility.

Histamine receptors rapidly desensitize without altering nerve-evoked contractions in murine urinary bladder smooth muscle.

Histamine has been implicated in urinary bladder dysfunction as an inflammatory mediator driving sensory nerve hypersensitivity. However, the direct influence of histamine on smooth muscle has not been thoroughly investigated. We hypothesized that histamine directly contracts urinary bladder smooth muscle (UBSM) independent of effects on nerves. Single cell quantitative RT-PCR determined that only histamine H1 and H2 receptors were expressed on UBSM cells. In isolated tissue bath experiments, histamine (200 µM) caused a highly variable and rapidly desensitizing contraction that was completely abolished by the H1 receptor antagonist fexofenadine (5 µM) and the Gq/11 inhibitor YM254890 (1 µM). Neither the muscarinic receptor antagonist atropine (1 µM), the Na+ channel blocker tetrodotoxin (1 µM), nor the transient receptor potential vanilloid type 1 antagonist capsazepine (10 µM) altered responses to histamine, suggesting that nerve activation was not involved. UBSM desensitization to histamine was not due to receptor internalization, as neither the cholesterol-depleting agent methyl-β-cyclodextrin (10 mM), the dynamin-mediated endocytosis inhibitor dynasore (100 µM), nor the clathrin-mediated endocytosis inhibitor pitstop2 (15 µM) augmented or prolonged histamine contractions. Buffer from desensitized tissues still contracted histamine-naïve tissues, revealing that histamine was not metabolized. Prolonged exposure to histamine also had no effect on contractions due to electrical field stimulation, suggesting that both efferent nerve and UBSM excitability were unchanged. Together, these data suggest that histamine, although able to transiently contract UBSM, does not have a lasting effect on UBSM excitability or responses to efferent nerve input. Thus, any acute effects of histamine directly on UBSM contractility are unlikely to alter urinary bladder function.NEW & NOTEWORTHY Histamine is commonly associated with inflammatory bladder pathologies. We sought to investigate the role of histamine on urinary bladder contractility. Histamine contracts the bladder, but this response is highly variable and desensitizes completely in minutes. This desensitization is not due to internalization of the receptor or metabolism of histamine. Because nerve-evoked contractions are also not increased in the presence of histamine, our findings suggest that histamine is not directly acting to change contractility.

K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

Decrease of urinary bladder contractility in patients with benign prostate hyperplasia

Aim of the study.Estimation of the decrease of urinary bladder contractility in patients with benign prostate hyperplasia (BPH).&#x0D; Patients and methods.In 146 BPH patients (mean age 62,1 0,8) urodynamic investigation including uroflowmetry and pressure-flow study was performed. The results of miction cystometry were evaluated using Shafer nomogram, the emptying idex was calculated as relation of voiding volume to maximal cystometric capacity.&#x0D; Results.56,2% BPH patients showed the decreased detrusor contractility. The results of urinary bladder contractility estimation by Shafer nomogram are dependent on the degree of infravesical obstruction (IVO). The emptying index is not related to the presence and degree of IVO and characterizes the degree of urinary bladder emptying as well as contraction duration. Concomitant lumbar osteochondrosis and diabetes detoriates urinary bladder emptying. Reliable contractility estimation may only be performed based on urodynamic study since basic BPH patients examination is not sufficient.&#x0D; Conclusion.In has to be considered during treatment strategy planning that in many BPH patients low urinary truct disfunction is determined by the decrease of urinary bladder contractility and not by IVO.

Effects of tadalafil versus silodosin on voiding function in male patients with non-neurogenic detrusor underactivity: A comparative study using propensity score matching.

To investigate and compare the effects of tadalafil and silodosin on lower urinary tract symptoms and voiding functions in men with non-neurogenic detrusor underactivity. A total of 126 treatment-naive men with lower urinary tract symptoms diagnosed as non-neurogenic detrusor underactivity received tadalafil (5mg/day) or silodosin (8mg/day) for 12months. After propensity score matching, parameter changes from before administration to 12months since treatment initiation were assessed based on subjective symptoms and urodynamic findings, including bladder contractility index and maximum urinary flow rate, and were compared between the tadalafil treatment group and the silodosin group. Detrusor underactivity was defined as bladder contractility index <100 and bladder outlet obstruction index <40. After propensity score matching, the final analysis included 48 patients each in the tadalafil and silodosin groups. No significant differences in prostate volume, subjective symptoms or urodynamic parameters were detected between the groups at baseline. Compared with baseline, significant improvements in subjective symptoms and storage and voiding functions were observed at month12 in both groups. Maximum urinary flow rate significantly improved by 1.7mL/s in the silodosin group and by 3.0mL/s in the tadalafil group. In addition, the mean bladder contractility index increased from 80.0 to 86.1 in the silodosin group and from 77.9 to 97.6 in the tadalafil group. Improvements in maximum urinary flow rate and bladder contractility index were significantly superior in the tadalafil group. Both tadalafil and silodosin significantly improve lower urinary tract symptoms and voiding function in patients with non-neurogenic detrusor underactivity. Furthermore, tadalafil is more effective than silodosin in improving bladder contractility index and maximum urinary flow rate.

Prostaglandin E2 and F2alpha Modulate Urinary Bladder Urothelium, Lamina Propria and Detrusor Contractility via the FP Receptor.

Current pharmacological treatment options for many bladder contractile dysfunctions are not suitable for all patients, thereby bringing interest to the investigation of therapies that target a combination of receptors. This study aimed to compare responses of PGE2 on the urinary bladder urothelium with lamina propria (U&LP, also called the bladder mucosa) or detrusor smooth muscle and attempt to identify the receptor subtypes mediating PGE2 contractile responses in these tissues. In the presence of selective EP1 – 4 receptor antagonists, varying concentrations of PGE2 were applied to isolated strips of porcine U&LP and detrusor that were mounted in organ baths filled with Krebs-bicarbonate solution and gassed with carbogen. The addition of PGE2 (1 and 10 μM) and PGF2α (10 μM) to U&LP preparations caused significant increases in the baseline tension and in the spontaneous phasic contractile frequency. In detrusor preparations, significant increases in the baseline tension were observed in response to PGE2 (1 and 10 μM) and PGFα (10 μM), and spontaneous phasic contractions were initiated in 83% of preparations. None of the selective PGE2 receptor antagonists inhibited the increases in baseline tension in both U&LP and detrusor. However, the antagonism of PGF2α receptor showed significantly inhibited contractile responses in both layers of the bladder. This study presents prostaglandin receptor systems as a potential regulator of urinary bladder contractility. The main contractile effects of PGE2 in both U&LP and detrusor are mediated via the FP receptor with no observed contribution from any of the four EP receptors.

Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells

Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin–actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Non-selective (HCN) Ion Channels Regulate Human and Murine Urinary Bladder Contractility.

Purpose: Hyperpolarization-activated cyclic nucleotide gated non-selective (HCN) channels have been demonstrated in the urinary bladder in various species. Since they play a major role in governing rhythmic activity in pacemaker cells like in the sinoatrial node, we explored the role of these channels in human and murine detrusor smooth muscle.Methods: In an organ bath, human and murine detrusor smooth muscle specimens were challenged with the HCN channel blocker ZD7288. In human tissue derived from macroscopically tumor-free cancer resections, the urothelium was removed. In addition, HCN1-deficient mice were used to identify the contribution of this particular isoform. Expression of HCN channels in the urinary bladder was analyzed using histological and ultrastructural analyses as well as quantitative reverse transcriptase polymerase chain reaction (RT-PCR).Results: We found that the HCN channel blocker ZD7288 (50 μM) both induced tonic contractions and increased phasic contraction amplitudes in human and murine detrusor specimens. While these responses were not sensitive to tetrodotoxin, they were significantly reduced by the gap junction inhibitor 18β-glycyrrhetic acid suggesting that HCN channels are located within the gap junction-interconnected smooth muscle cell network rather than on efferent nerve fibers. Immunohistochemistry suggested HCN channel expression on smooth muscle tissue, and immunoelectron microscopy confirmed the scattered presence of HCN2 on smooth muscle cell membranes. HCN channels seem to be down-regulated with aging, which is paralleled by an increasing effect of ZD7288 in aging detrusor tissue. Importantly, the anticonvulsant and HCN channel activator lamotrigine relaxed the detrusor which could be reversed by ZD7288.Conclusion: These findings demonstrate that HCN channels are functionally present and localized on smooth muscle cells of the urinary bladder. Given the age-dependent decline of these channels in humans, activation of HCN channels by compounds such as lamotrigine opens up the opportunity to combat detrusor hyperactivity in the elderly by drugs already approved for epilepsy.

The Long-Lasting Enhancing Effect of Distigmine on Acetylcholine-Induced Contraction of Guinea Pig Detrusor Smooth Muscle Correlates with Its Anticholinesterase Activity.

Distigmine bromide (distigmine), a reversible, long-lasting cholinesterase (ChE) inhibitor, is used for the treatment of underactive bladder in Japan and has been shown to potentiate urinary bladder (UB) contractility. We studied the duration of distigmine's potentiating effects on acetylcholine (ACh)-induced UB contraction and its inhibitory effects on ChE activity, and compared that with those of other ChE inhibitors (neostigmine, pyridostigmine, and ambenonium). The duration of potentiating/inhibitory effects of ChE inhibitors, including distigmine, on ACh-induced guinea pig UB contraction/ChE activity was evaluated for 12 h following washout. Dissociation rate constants (k) of the inhibitors were also tentatively calculated based on the time courses of their ChE inhibitory effects. The potentiating effect of distigmine (10-6 M) on ACh-induced UB contraction and its inhibitory effect on ChE activity were significantly sustained 12 h after washout. The potentiating effect of other ChE inhibitors on ACh-induced UB contraction, however, was sustained only until 3 h after washout. The ChE inhibitory effects of these inhibitors dissipated in a time-dependent manner after washout, with more than 75% of ChE activity restored by 4 h after washout. The k values of ChE inhibitors approached 0.50 h-1, except for distigmine, where k could not be determined. Compared with that of other ChE inhibitors, the potentiating effect of distigmine on UB contractile function was significantly more sustainable following washout, which was likely associated with its corresponding long-lasting ChE inhibitory effect. Distigmine may associate more strongly with UB ChE than other ChE inhibitors, which would partly explain its sustained effects.

Estimation of bladder contractility from intravesical pressure-volume measurements.

To describe parameters from urodynamic pressure recordings that describe urinary bladder contractility through the use of principles of muscle mechanics. Subtracted detrusor pressure and voided flow were recorded from patients undergoing filling cystometry. The isovolumetric increase of detrusor pressure, P, of a voluntary bladder contraction before voiding was used to generate a plot of (dP/dt)/P versus P. Extrapolation of the plot to the y-axis and the x-axis generated a contractility parameter, vCE (the maximum rate of pressure development) and the maximum isovolumetric pressure, P0 , respectively. Similar curves were obtained in ex vivo pig bladders with different concentrations of the inotropic agent carbachol and shown in a supplement. Values of vCE , but not P0 , diminished with age in female subjects. vCE was most significantly associated with the 20-80% duration of isovolumetric contraction t20-80 ; and a weaker association with maximum flow rate and BCI in women. P0 was not associated with any urodynamic variable in women, but in men was with t20-80 and isovolumetric pressure indices. The rate of isovolumetric subtracted detrusor pressure (t20-80 ) increase shows a very significant association with indices of bladder contractility as derived from a derived force-velocity curve. We propose that t20-80 is a detrusor contractility parameter (DCP). Neurourol. Urodynam. 36:1009-1014, 2017. © 2016 Wiley Periodicals, Inc.

Urinary Bladder Contractility Revisited. Correlation of Noninvasively and Invasively Measured Contractility Parameters in Patients Eligible for Transurethral Resection of the Prostate

To validate a noninvasively estimated measure of urinary bladder contractility by correlating it with 3 existing invasive contractility parameters and to compare and correlate those invasive parameters. A group of 74 patients, recruited in 3 different hospitals, and eligible for transurethral resection of the prostate on clinical grounds, were noninvasively studied preoperatively using the condom catheter method. The maximum condom pressure pcond.max measured during a mechanical interruption of flow rate was considered an estimate of urinary bladder contractility and compared to conventional contractility parameters calculated from preoperative (invasive) pressure-flow studies. The highest correlations were found between the invasive parameters. The correlation between the noninvasive parameter on the one hand and the invasive parameters on the other hand was lower, but mostly significant. In a number of patients, pcond.max underestimated the isovolumetric bladder pressure. The underestimated patients were more obstructed than those who were not underestimated and had a higher (invasively measured) contractility. When the underestimated patients were deselected, the correlation between the noninvasive pcond.max and the invasive parameters in the remaining 52 patients was higher. The 4 tested contractility parameters represent different aspects of urinary bladder contractility. Nevertheless, there was a significant correlation among them supporting the concept of a common basis, that is, detrusor contractility. The invasive contractility parameter bladder contractility index overestimated contractility in patients with lower urinary tract symptoms and/or benign prostatic enlargement. A modified parameter is suggested.

Local Change in Urinary Bladder Contractility Following CNS Dopamine Denervation in the 6-OHDA Rat Model of Parkinson's Disease.

Background: Urinary problems, including urinary frequency, urgency, and nocturia are some of the non-motor symptoms that correlate most with poor quality of life in Parkinson’s disease. However, the mechanism behind these symptoms is poorly understood, in particular regarding peripheral bladder pathophysiology following dopamine degeneration.Objective: In this study, we compared the contractile responsiveness of urinary bladder from the 6-OHDA unilateral rat model of Parkinson’s disease with that of normal untreated animals.Methods: The contractility of the urinary detrusor muscle was evaluated in bladder strip preparations using electrical field stimulation, and muscarinic and purinoceptor stimulations in an vitro organ bath setup.Results: Our data show that the overall contractile response following electrical field stimulation was significantly higher (43% at maximum contraction by 20–40 Hz stimulation) in the 6-OHDA-lesioned rats as compared to control animals. This increase was associated with a significant increase in the cholinergic contractile response, where the muscarinic agonist methacholine produced a 44% (at 10 −4 M concentration) higher response in the 6-OHDA-treated rats as compared to controls with a significant left-shift of the dose response. This indicates an altered sensitivity of the muscarinic receptor system following the specific central 6-OHDA-induced dopamine depletion. In addition a 36% larger contraction of strips from the 6-OHDA animals was also observed with purinoceptor activation using the agonist ATP (5×10 −3 M) during atropine treatment.Conclusions: Our data shows that it is not only the central dopamine control of the micturition reflex that is altered in Parkinson’s disease, but also the local contractile function of the urinary bladder. The current study draws attention to a mechanism of urinary dysfunction in Parkinson’s disease that has previously not been described.

Large conductance voltage‐ and Ca 2+ ‐activated K + channels mediate the 17β‐estradiol inhibitory effects on human urinary bladder smooth muscle cell excitability and contractility

Estrogens are critical regulators of smooth muscle function, including urinary bladder smooth muscle (UBSM) contractility. However, the mechanism underlying estrogen's regulatory role in UBSM has not been fully elucidated. Here, we explored whether 17β-estradiol,a physiologically active estrogen, activates large conductance voltage- and Ca2+-activated K+ (BK) channels in human UBSM. We obtained UBSM samples from 18 patients to examine the physiological role of 17β-estradiol in regulating UBSM function. 17β-estradiol inhibited UBSM spontaneous phasic contractions in a concentration-dependent manner (100 nM-1 µM). These 17β-estradiol inhibitory effects were prevented in the presence of paxilline (1 µM), a selective inhibitor of BK channels. 17β-estradiol (100 nM) also inhibited nerved-evoked contractions of UBSM isolated strips. Perforated patch-clamp data showed that 17β-estradiol increased the depolarization-induced whole cell outward currents, effects that were blocked by paxilline (1 µM). 17β-estradiol (100 nM) also increased the frequency of the transient BK currents in human UBSM cells. 17β-estradiol (100 nM) increased the BK channel open probability in inside-out excised patches from UBSM cells, supporting the concept that 17β-estradiol activates BK channels directly. Our results suggest that 17β-estradiol has a direct stimulatory effect on BK channel activity, thus decreasing the human UBSM excitability and contractility. Supported by NIH R01DK04284 to G.V. Petkov & NIH F31DK104528 to A. Provence.

The influence of doxazosin, an alpha1-adrenergic receptor antagonist on the urinary bladder contractility in pigs.

In the present study influence of doxazosin on the porcine urinary bladder contractility has been examined. Immature pigs were treated for 30 days with: a) doxazosin (n = 5) per os at a dose of 0.1 mg/kg b.w. or b) placebo (n = 5; control group). Thereafter, animals were sacrificed and urinary bladder strips from the trigone were suspended in organ baths. The tension of the smooth musce was measured before and after exposition to acetylocholine (ACh; 10(-5) - 10(-3) M), norepinephrine (NE; 10(-9) - 10(-7) M) and 5-hydroxytryptamine (5-HT; 10(-7) - 10(-5) M). Both the ACh and 5-HT at the highest doses significantly increased the contractility in each group, but this response was weaker in doxazosin-treated animals. NE caused relaxation in both groups, but the effect was weaker in doxazosine-treated group. The results of our study have shown that long-term administration of doxazosin caused a desensitization of the detrusor smooth muscle for in vitro applied mediators of the autonomic nervous systems.

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility (865.4)

Role of phosphodiesterase‐1 in muscarinic receptor‐induced human urinary bladder smooth muscle excitability and contractility Wenkuan Xin1, Ning Li1, Eric S. Rovner2, Qiuping Cheng1, and Georgi V. Petkov1,2 1Drug Discovery &amp; Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 and 2Medical University of South Carolina, Charleston, SC 29425 Activation of muscarinic M3 receptors (M3Rs) leads to an inhibition of the large conductance Ca2+‐activated K+ (BK) channels and an increase in human urinary bladder smooth muscle (UBSM) excitability. M3R‐mediated Ca2+ influx activates phosphodiesterase‐1 (PDE1) and reduces cellular cAMP levels. We investigated the mechanism by which the integrated PDE1 and MR signaling pathways stimulate human UBSM excitability and contractility. Pharmacological activation of M3Rs with carbachol (1 µM) decreased the frequency and amplitude of transient BK currents (TBKCs) in UBSM cells and subsequent inhibition of PDE1 with 8MM‐IBMX (10 µM) recovered TBKC activity. In the absence of M3R stimulation, inhibition of PDE1 increased TBKCs frequency, and subsequent inhibition of protein kinase A (PKA) with H‐89 (10 µM) reduced TBKCs frequency. 8MM‐IBMX (10 µM) hyperpolarized UBSM membrane potential, an effect which was blocked upon PKA inhibition. 8MM‐IBMX suppressed spontaneous phasic contractions and nerve‐evoked contractions of UBSM isolated strips. In conclusion, PDE1 enhances the M3R‐mediated inhibition of BK channels. Inhibition of PDE1 attenuates UBSM excitability by increasing BK channel activity via a PKA‐dependent mechanism. A combination of a PDE1 inhibitor and a MR antagonist can be an effective novel approach for the treatment of bladder overactivity.Key words: phosphodiesterase, muscarinic receptor, detrusorGrant Funding Source: NIH R01 DK084284 to Georgi V. Petkov

Changes of urinary bladder contractility in high‐fat diet‐fed mice: The role of tumor necrosis factor‐α

To study the role of tumor necrosis factor-α in bladder dysfunction associated with obesity. Male 8-week-old C57BL/6J mice were divided into three groups: (i) control mice; (ii) vehicle-treated high-fat diet-fed mice; and (iii) etanercept-treated high-fat diet-fed mice. High-fat diet feeding lasted for 12 weeks, vehicle or etanercept (0.8 mg/kg/day, a tumor necrosis factor-α antagonist) treatment was given during the last 4 weeks. At the end of the treatment period, serum tumor necrosis factor-α, total cholesterol, triglyceride and blood glucose were measured. Bladder strip contractile responses to 1 μmol/L acetylcholine or 50 mmol/L KCl were studied in an organ bath. Bladder protein kinase Cζ, nuclear factor-κB and intercellular adhesion molecule-1 expressions were analyzed using western blots. Serum levels of tumor necrosis factor-α total cholesterol, triglyceride and glucose were significantly elevated in high-fat diet-fed mice; and the levels were not ameliorated by etanercept treatment. High-fat diet-fed mouse bladder showed reduced contractile responses to acetylcholine and KCl stimulation accompanied by high expression levels of phospho-protein kinase Cζ, nuclear nuclear factor-κB and intercellular adhesion molecule-1. Etanercept restored normal bladder contractile responses, as well as protein kinase Cζ nuclear factor-κB and intercellular adhesion molecule-1 expressions. A high-fat diet induces bodyweight gain, hyperlipidemia and hyperglycemia in mice. Elevated serum tumor necrosis factor-α level associated with increased protein kinase Cζ phosphorylation, nuclear factor-κB nuclear migration, intercellular adhesion molecule-1 expression and impaired muscle contractility are shown in the high-fat diet-fed mouse bladder. Tumor necrosis factor-α antagonist treatment restores normal bladder contractility, and protein kinase Cζ nuclear factor-κB and intercellular adhesion molecule-1 levels.

Effect of hydrogen peroxide on contractility and citrate synthase activity of the rabbit urinary bladder in the presence and absence of resveratrol and a whole-grape suspension

One etiology related directly to obstructive urinary bladder dysfunction is ischemia/reperfusion resulting in significant oxidative stress to the bladder. Grapes, a natural source of antioxidants, have been proven effective in preventing obstructive and ischemic bladder dysfunction. Many investigators believe that resveratrol is the primary active antioxidant ingredient in grapes. We compared the ability of a whole-grape suspension with pure resveratrol in their ability to protect the bladder from in vitro oxidative stress mediated by hydrogen peroxide (H2O2). Four male rabbit bladders were used. Two strips from each bladder were incubated in the presence of 1 mg/mL grape suspension for 30 min, another two strips were incubated in the presence of 1 mg/mL resveratrol solution, and the last two strips were incubated in the presence of 1 mg/mL sucrose/and fructose as controls. The rest of the bladder was separated into muscle and mucosa, frozen and stored for biochemical evaluation. (1) Chemically, resveratrol has about 20 times the antioxidant capacity of the grape suspension. (2) The grape suspension had significant protective effects when the rate of tension was quantitated at all concentrations of H2O2, while the resveratrol had no effect. (3) Citrate synthase activities of the muscle and mucosa were significantly protected by the grape suspension but not by resveratrol. These data demonstrate that the grape suspension protects the mitochondria to a significantly greater degree than resveratrol, which suggests that the antioxidant activities are due to the combination of active components found in the grape suspension and not just resveratrol.

Effect of Vitis vinifera Leaf Hydroalcoholic Extract on Rat Urinary Bladder Contractility

Introduction: Several reports have shown the various medicinal effects of grape (Vitis vinifera) seed and skin extracts, such as antioxidant, hypotensive and vasodilatory effects. It has been recently shown the relaxatory effect of grape leaf hydroalcoholic extract on rat ileum, uterus, aorta, trachea and vas deferens as well as on frog isolated heart. The aim of the present study was to investigate the effect of Vitis vinifera leaf hydroalcoholic extract (VLHE) on contractility of urinary bladder smooth muscle in male rats and to study the mechanism(s) of its action. Materials and Methods: The extract of dried grape leaf was prepared by macerated method using 70% ethanol for 72h in room temperature. The solvent was then evaporated. Urinary bladder was removed from male adult wistar rats after induction of deep anaesthetize by di-ethyle-ether. Tissues were then suspended in the organ bath containing physiologic solution (37oC, pH 7.4) bubbled with oxygen. Contractions were recorded isometrically under 1g resting tension. Results: VLHE at 1, 2, 4 and 8 mg/ml significantly reduced the urinary bladder contractions evoked by KCl (60 mM P<0.05) and (0.5, 1, 2 and 4 mg/ml) also reduced the contractions evoked by Acetylcholine (5 µM) dose dependently (P<0.05). In calcium free physiologic solution, KCl-induced (120 mM P<0.05) contractions were occurred only after adding calcium (0.312, 0.625, 1.25, 2.5 and 5 mM) to the organ bath solution. VLHE (2 mg/ml) also reduced different concentration of calcium-induced contraction in the presence of KCl (P<0.05). The VLHE- induced urinary bladder relaxation was unaffected by propranolol (1 µM for 20 mins) while the presence of tetraethylamonium (10 mM for 20 mins) could strongly reduce the relaxatory effect of VLHE. Conclusion: The data suggest that VLHE induces spasmolytic effect in rat urinary bladder, possibly via blocking voltage dependent calcium channels and activation of calcium-activated potassium channels.