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PURPOSE: The urinary bladder generates phasic contractions via action potentials generated in pre- and then postganglionic neurons. Whilst the frequency-dependence of postganglionic neurons to generate contractions has been quantified, the dynamic range of preganglionic neurons is less clear and if intramural ganglia exert frequency-dependent modulation of transmission between pre- and postganglionic neurons. The phosphodiesterase type 5 inhibitor sildenafil reduces neurotransmitter release from postganglionic fibres to detrusor smooth muscle and an additional question was if there was also a preganglionic action. This study aimed to compare the frequency range of bladder contractile activation by pre- and postganglionic stimulation in pig and rat bladders and if sildenafil exerted additional preganglionic actions.METHODS: An arterially-perfused ex vivo pig bladder preparation was used for preganglionic (pelvic nerve) and mixed pre-and postganglionic (direct bladder wall) stimulation at 36°C and postganglionic mediated contractions achieved by field-stimulation of in vitro isolated detrusor strips. With rats, pelvic nerve stimulation was carried out in vivo and postganglionic stimulation also with isolated detrusor strips.RESULTS: All contractions were abolished by 2% lignocaine indicating they are nerve-mediated. Stimulation targets were verified with hexamethonium that completely abolished pelvic nerve responses by had no effect on detrusor strips; responses to mixed bladder wall stimulation were partially reduced. The frequency-dependence of contractile activation was similar whether by pre- or postganglionic stimulation in both pigs and rats. Sildenafil reduced contractions to preganglionic stimulation significantly more than to postganglionic stimulation. Mixed pre- and postganglionic stimulation were reduced by an intermediate extent.CONCLUSION: Intramural ganglia offer no frequency-dependent modulation under the experimental conditions used here and the sildenafil data are consistent with multiple sites of action underlying generation of bladder contractions. A translational aspect of these findings is discussed in terms of setting stimulation parameters for neuromodulation protocols.

BACKGROUND: Chronic ischemia is a known risk factor for the development of lower urinary tract symptoms (LUTS) and bladder hypocontractility. Less is known, however, about the impact of acute ischemia. Classic teaching suggests that collateral circulation is robust in the bladder and, therefore, loss of a single source of blood flow should have no deleterious effect. This study aims to provide visual evidence that segmental vascular supply is critical for maintaining adequate perfusion to the bladder. METHODS: Ex vivo pig bladders were cannulated bilaterally in the superior vesical arteries and perfused using contrasting red and green dye. Images were collected at each step of the perfusion dyeing process and these images were analyzed using a custom program to calculate the average hue of each side. Statistical analysis was performed using Student’s t-test. RESULTS: The two halves being perfused by separate arteries showed a statistically significant difference when compared (P

INTRODUCTION AND OBJECTIVE:We previously reported on the single slice T1 mapping of human bladder using a pair of spoiled gradient recalled echo (SPGR) images acquired at different flip angles. Her...

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

Recently, hydrogen sulfide (H2S) has been recognized as the third gasotransmitter besides nitric oxide and carbon monoxide, and it has been reported that H2S exhibits various physiological functions such as neuromodulation and vasorelaxation. In the lower urinary tract (bladder and prostate), it is reported that donors of H2S induce contraction of the rat detrusor and relaxation of the pig bladder neck. These reports suggest a possibility that H2S may have site-specific effects on the bladder. However, the detailed functions of H2S in each part of the bladder are still unclear. In addition, there is no report showing physiological roles of H2S in the prostate. In this article, we will review the distribution of enzymes related to H2S biosynthesis and physiological roles of H2S in the lower urinary tract based on reports from our and other groups. We will also introduce a possibility that H2S can be a new therapeutic target against lower urinary tract symptoms (LUTS) based on our data from spontaneously hypertensive rats (SHRs), which develop hypertension-mediated LUTS.

AIMS Chronic ischemia is a recognized factor in the pathophysiology of underactive bladder (UAB). Although relative ischemia (ie, low blood flow) is known to occur during filling, little is known regarding the pathophysiology that leads to UAB. Therefore, we developed an ex vivo functional porcine model to investigate the role of transient ischemia and whether autoregulation, a mechanism that maintains tissue oxygenation in certain vital organs, also exists in the bladder. METHODS Using bladders from slaughtered pigs, we prepared an isolated perfused model where we studied the effects of bladder perfusion flow rate on perfusion pressure and tissue oxygenation during the filling phase. Bladders were perfused at an initial flow rate of 20 mL/min and then clamped in a sequentially decreasing stepwise manner down to no flow and back to the initial flow rate. RESULTS We found a linear relationship between flow rate and perfusion pressure until the flow rate decreased below 5 mL/min at which point the vascular resistance decreased; however, tissue pO2 remained stable after an initial decline. CONCLUSIONS These findings suggest that there may be an intrinsic autoregulatory mechanism in the bladder that allows it to undergo cyclic episodes of relative ischemia during its normal function. Factors that overcome this mechanism such as complete or chronic ischemia may be critical in the progression to detrusor underactivity and thereby highlight the importance of intervention during the early phases of this disease process.

BMC urology 19(1), 62 (2019). doi:10.1186/s12894-019-0495-z, Published by BioMed Central, London

Many patients who suffer from spinal cord injuries (SCI) also suffer from neurogenic bladder dysfunction, and lack the sensation and control of their bladder. In order to alleviate the build up of bladder pressure from urine production and promote good renal health, it is recommended to perform clean intermittent catheterization (CIC) every 2 to 4 hours throughout the day. However, since urine production is not constant, sometimes the bladder will fill with urine to capacity before the recommended CIC time causing the patient to leak, adding unnecessary embarrassment. As such, incontinence is the primary concern of many SCI patients. Sadly, there are no practical solutions available on the market that addresses this concern. In this work, we investigate using near-infrared spectroscopy to develop a wearable and non-invasive bladder volume sensing system to provide timely alerts to SCI patients based on their current bladder volume. We showcase the feasibility of such a system using an optical phantom that mimics the bladder and by performing ex vivo measurements on a pig bladder and intestines.

Aims Although there is evidence that deficits in bladder blood flow negatively impact bladder function, the effects of vesical, and perfusion pressures on bladder perfusion (perfusate flow), and of perfusate flow on vesical pressure, remain poorly understood. The present study used the isolated perfused working pig bladder model to examine the relationships between blood flow, and vesical and perfusion pressures. Methods Vesical arteries of pig bladders obtained from a local slaughterhouse were cannulated and perfused with Krebs-Henseleit solution at different pressures, and with carbachol to cause bladder contraction. The urethra of each bladder was cannulated to permit filling (10 mL/min), isovolumetric contraction and emptying. A ureter was cannulated with a pressure sensor to monitor vesical pressure. Results When at rest (50 mL vesical volume), bladder vesical pressure was 8.06 ± 1.5 mmHg and perfusate flow driven by a pressure gradient of 105 mmHg was 22.5 ± 2 mL/min (58.9 ± 7.8 mL/min-100 g). During filling, vesical pressure increased and flow decreased, but not necessarily in-parallel. Perfusate flow decreased transiently during isovolumetric contraction, and flow increased during emptying. A reduction in perfusion pressure from ∼105 to ∼40 mmHg reduced flow from ∼70 to ∼20 mL/min-100g, and reduced flow correlated with reduced vesical pressure. Conclusion Perfusate flow is dependent on bladder perfusion pressure, and not necessarily reciprocally dependent on vesical pressure. Vesical pressure is highly sensitive to the level of perfusate flow, which supports the hypothesis that vesical pressure is dependent on the level of detrusor smooth muscle contractile activity (tone), and that compliance is dependent on bladder perfusion.

Because neuronal released endogenous H2S has a key role in relaxation of the bladder outflow region, we investigated the mechanisms involved in H2S dependent inhibitory neurotransmission to the pig bladder neck.Bladder neck strips were mounted in myographs for isometric force recording and simultaneous measurement of intracellular Ca(2+) and tension.On phenylephrine contracted preparations electrical field stimulation and the H2S donor GYY4137 evoked frequency and concentration dependent relaxation, which was reduced by desensitizing capsaicin sensitive primary afferents with capsaicin, and the blockade of adenosine 5'-triphosphate dependent K(+) channels, cyclooxygenase and cyclooxygenase-1 with glibenclamide, indomethacin and SC560, respectively. Inhibition of vanilloid, transient receptor potential A1, transient receptor potential vanilloid 1, vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide and calcitonin gene-related peptide receptors with capsazepine, HC030031, AMG9810, PACAP6-38 and CGRP8-37, respectively, also decreased electrical field stimulation and GYY4137 responses. H2S relaxation was not changed by guanylyl cyclase, protein kinase A, or Ca(2+) activated or voltage gated K(+) channel inhibitors. GYY4137 inhibited the contractions induced by phenylephrine and by K(+) enriched (80 mM) physiological saline solution. To a lesser extent it decreased the phenylephrine and K(+) induced increases in intracellular Ca(2+).H2S produces pig bladder neck relaxation via activation of adenosine 5'-triphosphate dependent K(+) channel and by smooth muscle intracellular Ca(2+) desensitization dependent mechanisms. H2S also promotes the release of sensory neuropeptides and cyclooxygenase-1 pathway derived prostanoids from capsaicin sensitive primary afferents via transient receptor potential A1, transient receptor potential vanilloid 1 and/or related ion channel activation.

Aims The current study investigates the role played by bradykinin (BK) receptors in the contractility to the pig bladder neck smooth muscle. Methods Bladder neck strips were mounted in myographs for isometric force recordings and BK receptors expression was also determined by immunohistochemistry. Results B2 receptor expression was observed in the muscular layer and urothelium whereas B1 expression was consistent detected in urothelium. A strong B2 immunoreactivity was also observed within nerve fibers among smooth muscle bundles. On urothelium-denuded preparations basal tone, BK induced concentration-dependent contractions which were reduced in urothelium-intact samples, by extracellular Ca2+ removal and by blockade of B2 receptors and voltage-gated Ca2+ (VOC) and non-VOC channels, and increased by cyclooxygenase (COX) inhibition. On phenylephrine-precontracted denuded strips, under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. In urothelium-intact samples, the B1 receptor agonist kallidin promoted concentration-dependent relaxations which were reduced by blockade of B1 receptors, COX, COX-1 and large-conductance Ca2+-activated K+ (BKCa) channels and abolished in urothelium-denuded samples and in K+-enriched physiological saline solution-precontracted strips. Conclusions These results suggest that BK produces contraction of pig bladder neck via smooth muscle B2 receptors coupled to extracellular Ca2+ entry via VOC and non-VOC channels with a minor role for intracellular Ca2+ mobilization. Facilitatory neuronal B2 receptors modulating NANC inhibitory neurotransmission and urothelial B1 receptors producing relaxation via the COX-1 pathway and BKCa channel opening are also demonstrated. Neurourol. Urodynam. 33:558–565, 2014. © 2013 Wiley Periodicals, Inc.

Recently, we have reported that docetaxel (DTX) loaded, amine terminated hyperbranched polyglycerol (HPG-C 8/10 -MePEG-NH 2 ) nanoparticles significantly increased drug uptake in mouse bladder tissues and was the most effective formulation to significantly inhibit tumor growth in an orthotopic model of bladder cancer. The objective of this study was to investigate the effects of HPG-C 8/10 -MePEG-NH 2 nanoparticles on bladder urothelial morphology and integrity, DTX uptake and permeability in bladder tissue and the extent of bladder urothelial recovery following exposure to, and then washout of, HPG-C 8/10 -MePEG-NH 2 nanoparticles. HPG-C 8/10 -MePEG-NH 2 nanoparticles significantly increased the uptake of DTX in both isolated pig bladder as well as in live mouse bladder tissues. Furthermore, HPG-C 8/10 -MePEG-NH 2 nanoparticles were demonstrated to increase the permeability of the urinary bladder wall by causing changes to the urothelial barrier function and morphology through opening of tight junctions and exfoliation of the superficial umbrella cells. These data suggest that exfoliation may be triggered by an apoptosis mechanism, which was followed by a rapid recovery of the urothelium within 24 h post-instillation of HPG-C 8/10 -MePEG-NH 2 nanoparticles. HPG-C 8/10 -MePEG-NH 2 nanoparticles cause significant but rapidly recoverable changes in the bladder urothelial morphology, which we believe may make them suitable for increasing drug permeability of bladder tissue and intravesical drug delivery.

Aims There is no information about the signaling pathways involved in the endothelin-1 (ET-1)-induced contraction of bladder neck. The current study investigates the mechanisms involved in the ET-1-elicited contraction in the pig bladder neck. Methods Bladder neck strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O2 and 5% CO2, for isometric force recording to endothelin receptor agonists, noradrenaline (NA), and electrical field stimulation. Endothelin ETA receptor expression was also determined, by both immunohistochemistry and Western blot. Results ETA receptor expression (Western blot) was observed in the muscular layer and urothelium. A strong ETA-immunoreactivity (ETA-IR) was identified within nerve fibers among smooth muscle bundles. ET-1 and ET-2 evoked similar concentration-dependent contractions of urothelium-denuded preparations. ET-3 produced a slight response, whereas the ETB receptor agonist BQ3020 failed to promote contraction. BMS182874, an ETA receptor antagonist, reduced ET-1-induced contraction whereas BQ788, an ETB antagonist, did not change such responses. ET-1 contractions were reduced by extracellular Ca2+ removal and by inhibition of voltage-gated Ca2+ (VOC) (L-type) and non-VOC channels, Rho/Rho-kinase pathway, and neuronal VOC channels. NA produced contractions which were enhanced by ET-1 threshold concentrations. ETA receptor blockade enhanced nitric oxide-dependent nerve-mediated relaxations. Conclusions These results suggest that ET-1 produces contraction via muscular ETA receptors coupled to extracellular Ca2+ entry via VOC (L-type) and non-VOC channels. Intracellular Ca2+ mobilization and a Rho/Rho-kinase pathway could also be involved in these responses. ET-1-evoked potentiation on noradrenergic contraction, and neuronal ETA receptors modulating nitrergic inhibitory neurotransmission, are also demonstrated. Neurourol. Urodynam. 31:156–161, 2012. © 2011 Wiley Periodicals, Inc.

We studied the role of calcitonin gene-related peptide in nonadrenergic, noncholinergic neurotransmission to the pig bladder neck.We used immunohistochemical techniques to determine the distribution of calcitonin gene-related peptide immunoreactive fibers as well as organ baths for isometric force recording. We investigated relaxation due to endogenously released or exogenously applied calcitonin gene-related peptide in urothelium denuded phenylephrine precontracted strips treated with guanethidine, atropine and NG-nitro-L-arginine to block noradrenergic neurotransmission, muscarinic receptors and nitric oxide synthase, respectively.Rich calcitonin gene-related peptide immunoreactive innervation was found penetrating through the adventitia and distributed in the suburothelial and muscle layers. Numerous, variable size, varicose calcitonin gene-related peptide immunopositive terminals were seen close below the urothelium. In the muscle layer calcitonin gene-related peptide immunopositive nerves usually appeared as varicose terminals running along muscle fibers. Electrical field stimulation (2 to 16 Hz) and exogenous calcitonin gene-related peptide (0.1 nM to 0.3 μM) evoked frequency and concentration dependent relaxation, respectively. Nerve responses were potentiated by capsaicin, decreased by calcitonin gene-related peptide (8-37) and abolished by tetrodotoxin, capsaicin sensitive primary afferent blockers, calcitonin gene-related peptide receptors and neuronal voltage gated Na+ channels. Calcitonin gene-related peptide-induced relaxation was potentiated by the neuronal voltage gated Ca2+ channels blocker ω-conotoxin-GVIA and decreased by calcitonin gene-related peptide (8-37). Calcitonin gene-related peptide relaxation was not modified by blockade of endopeptidases, nitric oxide synthase, guanylyl cyclase and cyclooxygenase.Results suggest that calcitonin gene-related peptide is involved in the nonadrenergic, noncholinergic inhibitory neurotransmission of the pig bladder neck, producing relaxation through neuronal and muscle calcitonin gene-related peptide receptors. Nitric oxide/cyclic guanosine monophosphate and cyclooxygenase pathways do not seem to be involved in such responses.

OBJECTIVE • To study the pharmacokinetic and toxicity profile of intravesically administered TMX-101, with its active ingredient R-837, a synthetic Toll-like receptor (TLR)-7 agonist, in a pig model. MATERIALS AND METHODS • TLR-7 expression was determined by immunohistochemistry in human and pig bladder tissue. • Four groups of six pigs received a 1-h intravesical instillation with R-837 of different formulations. • Pharmacokinetic analysis was performed on plasma. Toxicity evaluation included monitoring the well-being of the animals, peripheral blood cell counts, and interleukin-6 and creatinine measurements. Urine was collected for R-837 measurement and dipstick analysis. • In total, three pigs per group were sacrificed 24 h post-treatment, and the remaining animals were sacrificed after 1 week. Histopathological examination of the bladder wall was performed. RESULTS • TLR-7 was homogeneously expressed in human and pig urothelium. • R-837 and vehicle were well tolerated without deterioration in animal well-being. • Systemic R-837 absorption was low. • Mean maximum plasma concentration of R-837 differed depending on the formulation. Post-treatment, plasma levels were negligible at 24 h. • Histopathological examination of the bladders did not show significant abnormalities, apart from the intended inflammatory reaction in the R-837 treated groups. CONCLUSION • Intravesically administered R-837 in pigs, which showed a similar TLR-7 distribution in bladder tissue as humans, is well tolerated and causes no bladder wall toxicity, and formulations with poloxamer and hydroxypropyl-β-cyclodextrin showed less systemic absorption.

Aims: Relaxation of detrusor muscle via beta-adrenoceptors may contribute to urine storage during bladder filling. Thus there is increasing interest in beta-adrenoceptor agonists as a potential treatment for detrusor overactivity. The role of the urothelium in bladder responses to beta-adrenoceptor agonists is not yet clear, although we have shown that these agonists have a greater inhibitory effect on detrusor contraction when the urothelium is intact. The aim was to determine which beta-adrenoceptor subtype is involved in this effect. Methods: Paired strips of pig bladder dome mucosa-intact and mucosa-denuded, were mounted in tissue baths. Relaxation responses were obtained to beta-adrenoceptor agonists (isoprenaline, dobutamine, salbutamol or BRL37344) in carbachol pre-contracted tissues. Inhibitory effects were studied by obtaining concentration-response curves (CRCs) to carbachol in the presence and absence of beta-adrenoceptor agonists. The inhibitory effects of isoprenaline were also studied following incubation with beta-adrenoceptor antagonists (propranolol, CGP20712, ICI-118, 551 or SR 59230A; non selected, beta(1), beta(2) and beta(3) selective respectively). Results: isoprenaline, dobutamine, salbutamol and BRL37344 all relaxed carbachol pre-contracted tissues and responses were similar in intact and denuded strips. In inhibition experiments, padrenoceptor agonists caused rightward shifts of carbachol CRCs. In intact strips the shift was greater with isoprenaline and BRL37344, but not with dobutamine or salbutamol. This increased shift was still observed in tissues pre-incubated with propranolo, CGP20712 and ICI-118, 551, but not with SR 59230A. Conclusions: beta(3)-adrenoceptors are involved in mediating inhibitory effects of beta-adrenoceptor agonists on detrusor contractions via the urothelium in pig bladder dome.

Objectives Bladder outlet obstruction is correlated with an increased peroxidation of lipids. The aldehyde 4-hydroxynonenal (HNE) is produced in relative large amounts during lipid peroxidation. The aim of this study was to investigate the effect of HNE on excitation-contraction coupling of detrusor smooth muscle. Methods We used smooth muscle strips from pig urinary bladder. Contractile responses to electrical field stimulation (EFS) (4 to 32 Hz), carbachol (10 −8 to 3.10 –5 M), and potassium (65.3 mM) were monitored before and after the addition of HNE. We investigated the effect of the synthetic thiol inactivator N -ethylmaleimide (NEM) on the stimulation pathways and compared it with the HNE-mediated effect. Results Responses of detrusor strips to EFS were for the greater part based on neurogenic stimulation and the release of acetylcholine. HNE (100 μM) diminished contractile responses to EFS and carbachol to the same extent. The pD 2 value of the carbachol concentration response curve did not decrease after exposure to HNE. The maximal effect obtained with carbachol was significantly reduced after 100 μM HNE treatment. Contractions induced by potassium were affected in a similar degree by HNE compared with EFS- and carbachol-induced responses of comparable amplitude. Incubation of bladder strips with NEM had similar effects on pharmacological responses compared with HNE exposure. Conclusions 4-Hydroxynonenal affects pig bladder contractility. L-type calcium channels and or the contractile system of the bladder muscle are susceptible to HNE-mediated damage. The cholinergic nerves and the muscarinic receptor signaling system remain largely unaffected. The effects of HNE are most likely mediated via alkylation of sulfhydryl groups.

Aims: Several pathophysiological conditions in the urinary bladder, for example, ischemia/reperfusion and in£ammation are characterized by the formation of reactive oxygen species (ROS). The ROS are highly toxic because they can destroy proteins, DNA, and lipids. The aim of this study was to investigate the eiect of oxidative stress on excitation-contraction coupling of detrusor smooth muscle. Materials and Methods: Smooth muscle strips were dissected from pig urinary bladder and mounted in organ baths. Oxidative stress was mimicked by the addition of Cumene hydroperoxide (CHP), a lipophilic hydroperoxide, to the organ baths. Contractile responses to electrical ¢eld stimulation (EFS: 4 ^32 Hz), carbachol (10 � 8 ^3� 10 � 5 M), potassium (65.3 mM), and ATP (1 mM) were monitored before and after the addition of CHP. Results: Responses of detrusor strips to EFS were for the greater part based on neurogenic stimulation and the release of acetylcholine. CHP diminished contractile responses to EFS and carbachol to the same extent. The pD2 value of the carbachol concentration-response curve decreased signi¢cantly after exposure to 0.1 mM, 0.4 mM, 0.8 mM CHP. Furthermore the maximal eiect obtained with carbachol was signi¢cantly reduced after 0.1 mM, 0.4 mM, and 0.8 mM CHP treatment. Contractions induced by potassium and ATP were signi¢cantly less aiected by oxidative stress compared to EFS- and carbachol-induced responses of comparable amplitude. Conclusions: The results of our study demonstrate that oxidative stress induced by CHP aiects pig bladder contractility. The muscarinic receptor signaling system is severely damaged. L-type calcium channels and the contractile system are less aiected and cholinergic nerves remain largely unaiected. Neurourol. Urodynam. 26:302 ^308, 2007. 2006 Wiley-Liss, Inc.

Authors from Sheffield and the USA investigated the role of neurokinin receptors in mediating contraction of detrusor muscle strips from human and pig. They found that NK-2 receptors subtypes mediate neurokinin A-induced contraction of the detrusor muscle, and that the NK-3 receptor appears not to have such a role. Another study, from a variety of European and American centres, determined the expression and plasma membrane location of isoforms of Na,K-ATPase in the rat ventral prostate; they were localised in the lateral and basolateral plasma membrane domain prostatic epithelial cells, and the authors draw conclusions about the significance of these findings. Finally authors from Yale University investigated the effects of castration on the expressions of endothelins, endothelin receptors and converting enzyme-1 in the rat seminal vesicle. Their findings led them to suggest that testosterone has a regulatory role in the expression of the endothelin receptor system in the rat seminal vesicle. OBJECTIVE To investigate the role of neurokinin (NK)-2 and -3 receptors in mediating the contraction of detrusor muscle strips from human and pig, to determine whether the pig is a good model for the study of tachykinin receptors in the human bladder, as the biological actions of tachykinins, e.g. substance P and NKA are mediated via three distinct receptor subtypes, NK-1, -2 and -3. MATERIALS AND METHODS Strips of detrusor muscle were obtained from the bladder dome and neck of female pigs and from patients undergoing cystectomy. Cumulative concentration-response curves to NKA were obtained in the absence and presence of either the NK-2 receptor-selective antagonist SR48968 or the NK-3 receptor-selective antagonist SB223412. RESULTS NKA produced concentration-dependent contractions in the human and pig detrusor muscle; the curves were shifted to the right by SR48968, with high affinity (pKB 8.9, 8.3 and 8.0 in the human, pig dome and pig neck, respectively), whereas SB223412 had a minimal effect (pKB 5.8, 5.8 and 6.3, respectively). CONCLUSION These data confirm that the NK-2 receptor subtype mediates NKA-induced contraction of the human and pig detrusor muscle. The NK-3 receptor appears to have no role in detrusor contraction of either species. The results also provide evidence that the NK-2 receptor in human and pig are the same, and the latter may be an appropriate species to study tachykinin-induced contractions in human bladder.

Aims To investigate the role of β-adrenoceptor subtypes in mediating relaxation of the pig bladder trigonal muscle in vitro. Materials and Methods Longitudinal strips of trigonal muscle were isolated, and the mucosa and serosa removed. Tissues were precontracted with KCl, and β-adrenoceptor agonists (isoprenaline or salbutamol) were added cumulatively, and concentration–relaxation curves (CRCs) were obtained. CRCs to agonists were obtained in the absence and presence of antagonists and antagonist affinity values were calculated. Results Isoprenaline (non-selective β-agonist) relaxed with high potency (pEC50 = 7.2). Propranolol antagonized CRCs to isoprenaline with a high affinity (apparent pKB = 8.8), but the Schild plot had a slope significantly (P

Using our model to grow superficial human bladder cancer in the mouse bladder, we have found that the polyamide compound, Syn3, when injected intravesically for 1 hour at 1 mg/mL on two consecutive days, markedly increases rAd-beta-gal intravesical gene transfer and expression. This enhanced transgene expression was much greater than obtain by the use of 22% ethanol, which had previously been shown to increase intravesical adenoviral gene transfer, whereas little or no gene expression was seen with exposure to only rAd-beta-gal. beta-Galactosidase staining was seen in virtually every normal urothelial and superficial tumor cell present, including tumors that express little or no coxsackie-adenovirus receptors when Syn3 was present. High adenoviral-mediated gene transfer was also documented in the pig bladder using Syn3 in a similar protocol. Therefore, Syn3 may overcome the limitations of adequate intravesical adenoviral-mediated gene transfer and, when combined with an appropriate adenoviral-mediated gene, could offer an effective approach to the treatment of superficial bladder cancer and perhaps even genetically altered precursor lesions.

Objective To determine whether detrusor muscle from the pig can be used as a model to study presynaptic 5-hydroxytryptamine (HT)4-receptor-mediated effects on the electrical field stimulated (EFS) cholinergic responses previously identified in the human bladder. Materials and methods Strips of detrusor muscle were mounted in physiological Krebs' solution under 1 g tension and field stimulated (25 Hz, 0.01 ms duration, 60 V for 5 s) at 100-s intervals and allowed to equilibrate. Concentration-response curves to 5-HT (1 nmol/L to 300 µmol/L) were constructed in the presence and absence of the 5-HT4-selective antagonists RS-100235 and GR-113808 (both at 0.3, 1 and 3 nmol/L). All experiments were conducted in the presence of methiothepin and ondansetron (both 1 µmol/L) to block 5-HT1, -HT2 and -HT3 receptors. Results 5-HT potentiated the cholinergic responses to EFS in pig bladder strips in a concentration-dependent manner, with a maximum mean ( sem) potentiation of 48.3 (7.7)% of the resting tension (n = 25). BothRS-100235 and GR-113808 antagonized the effect of 5-HT with high affinity, yielding apparent pKB values which were consistent with the responses being mediated via the 5-HT4 receptor subtype. Conclusion These data indicate that 5-HT can potentiate EFS responses in isolated pig bladder strips and that the 5-HT4 receptor subtype mediates this response. Therefore, the pig may be used as an effective model to study presynaptic 5-HT4 receptors previously reported in the human bladder.

Purpose: To investigate whether the pig is a suitable model for studies of lower urinary tract function and dysfunction, we sought to determine the morphology of the female pig bladder neck and urethra. Computer assisted 3-dimensional (D) reconstructions from step serial histological sections were used for visualization of the spatial relationships between neighboring urethral wall components, and the quantification of these components in the bladder neck and along the urethra.Materials and Methods: Step serial histological paraffin sections from the bladder neck and urethra of 6 female pigs, stained with Masson’s trichrome, were used to generate computer assisted 3-D reconstructions using MacStereology (Ranfurly MicroSystems Ltd., Airdrie, United Kingdom) as the 3-D software package.Results: The bladder neck and urethral anatomy revealed well defined smooth and striated muscle layers that varied in location, regional distribution and orientation. Circular smooth muscle was maximally developed in the mid ...

Objective To investigate the effect of partial bladder outlet obstruction on detrusor blood flow and oxygen tension (PdetO2) in female pigs. Materials and methods Detrusor-layer oxygen tension and blood flow were measured using oxygen-sensitive electrode and radiolabelled microsphere techniques in five female Large White pigs with a partial urethral obstruction and in five sham-operated controls. The effects of chronic outlet obstruction on bladder weight, and cholinergic nerve density and distribution, are also described. Results In the obstructed bladders, blood flow and oxygen tension were, respectively, 54.9% and 74.3% of control values at low bladder volume, and 47.5% and 42.5% at cystometric capacity. Detrusor blood flow declined by 27.8% and 37.5% in the control and obstructed bladders, respectively, as a result of bladder filling, whilst PdetO2 did not decrease in the controls, but fell by 42.7% in the obstructed bladders. Bladder weight increased whilst cholinergic nerve density decreased in the obstructed animals. Conclusion In pigs with chronic bladder outlet obstruction, blood flow and oxygen tension in the detrusor layer were lower than in control animals. In addition, increasing detrusor pressure during filling caused significantly greater decreases in blood flow and oxygen tension in the obstructed than in the control bladders.

Objective To investigate the role of cholinergic excitation in mediating changes in detrusor compliance (manifested on conventional cystometry as an incremental rise in detrusor pressure as bladder volume increases) under conditions of propofol-sedation in the pig. Materials and methods Consecutive cystometrograms (CMGs) were obtained from eight female Large White pigs at a bladder filling rate of 50 mL/min. The first CMG was obtained while the pig was awake and unsedated. Two subsequent CMGs were obtained after light to moderate sedation with propofol (2–8 mg/kg/h) before and after the administration of intravenous atropine (0.02 mg/kg). Results All bladders were highly compliant over the volumes instilled (before sedation) with a maximum pressure during the filling phase of 0.9 cmH2O and a compliance of 943 mL/cmH2O. After sedation with propofol, the maximum pressure during the filling phase increased to 14 cmH2O with a compliance of 69 mL/cmH2O. Atropine antagonized this change in compliance; after sedation and atropine, the maximum pressure during the filling phase decreased to 4 cmH2O (P

The aim of our study was to analyze the physical and histological behavior of a second generation collagen (GAX 65*) in the mini-pig bladder, comparing it to the commonly used substance for injection (GAX 35*). GAX 65 is a glutaraldehyde cross-linked bovine collagen with a collagen concentration of 65 mg./ml. This much higher collagen concentration could be achieved due to sophisticated manufacturing technology. In 18 mini-pigs 2 to 4, 1 ml. deposits of GAX 65 and 35, respectively, were implanted into the submucosal space of the surgically opened bladder. The animals were sacrificed at months 1, 2, 4 and 6. The elevation (height equals protrusion of the implant into the bladder) and volume of the implants were measured and calculated immediately after injection and at necropsy. Histology was performed. The GAX 65 implants had a lesser decrease of elevation and volume during the 6-month observation period. Histology showed that the invasion of endogenous fibroblasts into the GAX 35 and 65 implants, and the formation of porcine collagen types I and III were almost identical. Clinically GAX 65 has better elevation properties than GAX 35 although statistical analysis did not show any significant difference (p = 0.07 to 0.25). However, preservation of the volume of the GAX 65 implant was significantly superior (por = 0.05) compared to that of GAX 35. Therefore, GAX 65 will most probably prove to be an excellent substance for the endoscopic treatment of vesicoureteral reflux and incontinence in children.

We investigated the possible involvement of H2S in nitric oxide independent inhibitory neurotransmission to the pig bladder neck.We used immunohistochemistry to determine the expression of the H2S synthesis enzymes cystathionine γ-lyase and cystathionine β-synthase. We also used electrical field stimulation and myographs for isometric force recordings to study relaxation in response to endogenously released or exogenously applied H2S in urothelium denuded, phenylephrine precontracted bladder neck strips under noradrenergic, noncholinergic, nonnitrergic conditions.Cystathionine γ-lyase and cystathionine β-synthase expression was observed in nerve fibers in the smooth muscle layer. Cystathionine γ-lyase and cystathionine β-synthase immunoreactive fibers were also identified around the small arteries supplying the bladder neck. Electrical field stimulation (2 to 16 Hz) evoked frequency dependent relaxation, which was decreased by DL-propargylglycine and abolished by tetrodotoxin (blockers of cystathionine γ-lyase and neuronal voltage gated Na(+) channels, respectively). The cystathionine β-synthase inhibitor O-(carboxymethyl)hydroxylamine did not change nerve mediated responses. The H2S donor GYY4137 (0.1 nM to 10 μM) induced potent, concentration dependent relaxation, which was not modified by neuronal voltage gated Na(+) channels, or cystathionine γ-lyase or cystathionine β-synthase blockade.Results suggest that endogenous H2S synthesized by cystathionine γ-lyase and released from intramural nerves acts as a powerful signaling molecule in nitric oxide independent inhibitory transmission to the pig bladder neck.

Characterizing the integrative physiology of the bladder requires whole organ preparations. The purpose of this study was to validate an isolated large animal (pig) bladder preparation, through arterial and intravesical drug administration, intravesical pressure recording, and filming of surface micromotions. Female pig bladders were obtained from the local abattoir and arterially perfused in vitro. Arterial and intravesical pressures were recorded at varying volumes. Bladder viability was assessed histologically and by monitoring inflow and outflow pH. Arterial drug administration employed boluses introduced into the perfusate. Intravesical administration involved slow instillation and a prolonged dwell-time. Surface micromotions were recorded by filming the separation of surface markers concurrently with intravesical pressure measurement. Adequate perfusion to all bladder layers was achieved for up to 8 h; there was no structural deterioration nor alteration in inflow and effluent perfusate pH. Arterial drug administration (carbachol and potassium chloride) showed consistent dose-dependent responses. Localized movements (micromotions) occurred over the bladder surface, with variable correlation with fluctuations of intravesical pressure. The isolated pig bladder is a valid approach to study integrative bladder physiology. It remains viable when perfused in vitro, responds to different routes of drug administration and provides a model to correlate movements of the bladder wall directly to variation of intravesical pressure.

The objective of this randomized in-vitro study was to compare the suturation time, integrity, and quality of the bladder closure in fresh cadaver pig bladders performed with barbed polyglyconate sutures vs polyglactin 910 sutures in running and interrupted fashion.Forty-eight pig bladders, each weighing from 120 to 150 g, were randomly divided into three groups: Group 1 (interrupted polyglactin 910 suture group), group 2 (running polyglactin 910 suture group), and group 3 (running barbed polyglyconate suture group). The bladder defects were closed laparoscopically, and the suturation times were noted. Two surgeons evaluated the integrity of each bladder closure. A cystometry was performed, and the filling and leak pressures were noted. A Kruskal-Wallis variance analysis test was used to compare the results of the three groups, and P0.05 was considered significant.There was a statistically significant difference between the mean suturation times of three groups: Group 1, 15.2 minutes; group 2, 9.14 minutes; and group 3, 7.13 minutes (P0.05). Mean bladder capacity at the time of leakage was 276.2, 353.8, and 419.7 mL for groups 1, 2, and 3, respectively, and the difference was statistically significant (P0.05).For the first time, we demonstrated laparoscopic knotless closure of bladder defects using the barbed polyglyconate suture material in an experimental in-vitro model. Closing the pig bladder with running knotless barbed suture provides a more effective and faster watertight bladder closure than traditional polyglactin 910 suture material.

Aims The involvement of endothelin receptors in the contraction of the lower urinary tract smooth muscle is well established. There is scarce information, however, about endothelin receptors mediating relaxation of the bladder outlet region. The current study investigates the possible existence of endothelin ETB receptors involved in the relaxation of pig bladder neck. Methods ETB receptor expression was determined by immunohistochemistry and urothelium-denuded bladder neck strips were mounted in organ baths for isometric force recording. Results ETB-immunoreactivity (ETB-IR) was observed within nerve fibers among smooth muscle bundles and urothelium. BQ3020 (0.01–300 nM), an ETB receptor agonist, produced concentration-dependent relaxations which were reduced by BQ788, an ETB receptor antagonist, and by inhibitors of protein kinase A (PKA) and large (BKCa)- or small (SKCa)-conductance Ca2+-activated K+ channels. Pretreatment with BKCa or SKCa channel inhibitors plus PKA blocking did not cause further inhibition compared with that exerted by inhibiting BKCa or SKCa channels only. BQ3020-induced relaxation was not modified by blockade of either nitric oxide (NO) synthase, guanylyl cyclase, cyclooxygenase (COX) or of intermediate-conductance Ca2+-activated-(IKCa), ATP-dependent-(KATP), or voltage-gated-(Kv) K+ channels. Under non-adrenergic non-cholinergic (NANC) conditions, electrical field stimulation (0.5–16 Hz) evoked frequency-dependent relaxations, which were reduced by BQ788 and potentiated by threshold concentrations of BQ3020. Conclusions These results suggest that BQ3020 produces relaxation of the pig bladder neck via activation of muscle endothelin ETB receptors, NO/cGMP- and COX-independent-, cAMP–PKA pathway-dependent-mechanisms, and involving BKCa and SKCa channel activation. ETB receptors are also involved in the NANC inhibitory neurotransmission. Neurourol. Urodynam. 31:688–694, 2012. © 2012 Wiley Periodicals, Inc.

To investigate the effects of fasudil, a Rho-associated serine-threonine protein kinase inhibitor, on contraction of the pig urinary bladder tissues with or without urothelium.Cumulative concentration-response curves (CRCs) to carbachol were obtained with and without 3-10 microM fasudil. Drug effects were evaluated in detrusor with and without urothelium. Inhibitory responses to fasudil were also examined in tissues precontracted with KCl and carbachol, and in response to electrical field stimulation, in pig bladder with and without urothelium.In detrusor without urothelium, maximum contraction (E(max)) decreased after administration of fasudil at 3 or 10 micromol/L (both P0.01), or 30 micromol/L (72.5 + or - 7.43%, 58.4 + or - 8.04% and 68.4 + or - 9.6%, respectively, of the first curve). In detrusor with urothelium, E(max) decreased significantly (all P0.05) after the addition of 3, 10 or 30 micromol/L of fasudil (84.9 + or - 6.7%, 67.9 + or - 5.2% and 35.2 + or - 4.1%, respectively). In tissues precontracted with 80 mmol/L KCl or 100 micromol/L carbachol, tension after administration of fasudil (1 nmol/L to 100 micromol/L) decreased (by approximately 40%), only after administration of fasudil at high concentration (1 micromol/L), in detrusor both with and without urothelium. In tissues with and without urothelium, responses to electrical field stimulation at 1-50 Hz decreased significantly in a concentration-dependent manner after addition of fasudil (3 to 30 micromol/L).Fasudil seems to provoke relaxation of the bladder detrusor via both urothelium-dependent and independent pathways.

To present a newly developed, low-cost, easy-to-use training model for diagnostic and therapeutic procedures in bladder pathology with real-time haptics.The model consists of a white plastic box in which a removable unit is placed and has been constructed to accommodate a prepared pig bladder on a metal plate. Small and large bladder tumors can be created in the bladder enabling diagnostics and treatment. Also, foreign bodies, such as bladder stones, can be inserted for training in cystolitholapaxy.This training model provides a realistic environment for training with the same instruments that are used in clinical practice. Numerous procedures can be performed, such as flexible and rigid urethrocystoscopy, biopsy taking, transurethral tumor resection, and cystolitholapaxy. The model is cheap and simple to create and install. Its practical usefulness has been proven during several training courses. Validation studies are under way.Training in diagnostic and therapeutic procedures in bladder pathology can be performed using this novel pig bladder model. The model offers a nonpressurized environment in which residents can acquire practical as well as procedural skills and material knowledge of urethrocystoscopy, transurethral resection of bladder tumor, biopsy taking, and cystolitholapaxy.

OBJECTIVE To investigate the influence of the mucosa on the inhibitory effects of the ATP-sensitive potassium channel (KATP channel) opener, cromakalim, on the spontaneous contractions of pig bladder strips from the bladder dome and trigone. Little is known about the influence of the mucosa on spontaneous contractions and whether the nature of these contractions differs between the bladder dome and trigone. MATERIALS AND METHODS Paired longitudinal strips of female pig bladders were isolated from the dome and trigone. The mucosa was removed from one strip per pair and tissues were set up in organ baths. Spontaneous activity was allowed to develop and recorded, and then cumulative concentration–response curves to cromakalim were obtained. The time needed for spontaneous contractions to develop, the frequency and amplitude of spontaneous contractions, and the effect of cromakalim were analysed. The strips of mucosa removed from the dome to produce denuded strips were also analysed by immunofluorescence using antibodies specific for vimentin and α-smooth muscle actin (α-SMA). RESULTS In the dome removal of the mucosa delayed the development of spontaneous contractions compared with mucosa-intact strips, whilst the trigone strips developed spontaneous contractions soon after set up in the organ baths irrespective of the presence or absence of mucosa. In the dome, cromakalim was more potent in suppressing spontaneous contractions when the mucosa was absent; whilst in the trigone the effects of cromakalim were similar in mucosa-intact and denuded strips. Upon examination of the strips of mucosa by immunofluorescence these strips were shown to contain cells positive for α-SMA or vimentin and cells positive for both, suggesting the presence of not only urothelium but also suburothelium and some detrusor smooth muscle bundles. CONCLUSION In the dome, the urothelium and suburothelium reduce the inhibitory effect of cromakalim on spontaneous contractions, whilst in the trigone these structures appear to have little influence. The mechanism for generating spontaneous contractions in the intact strips seems to be linked to the urothelium and suburothelium in the dome but not in the trigone.

Aims The objective of the study was to establish an experimental model for the extracorporeal perfusion of the pig detrusor. In order to validate this model we examined some biochemical parameters and determined the effect of carbachol on the contractility of perfused female pig bladders. Methods Twenty-six pig bladders were perfused with Krebs–Ringer bicarbonate-glucose buffer for a period up to 5 hr with the aim to preserve a viable organ, which would be responsive to contraction-inducing agents. The intravesical pressure of the bladder as well as the intraarterial pressure of the vesical arteries were recorded before and after administration of carbachol. Results The perfusate pH, lactate, partial carbon dioxide tension, and the ATP content in the perfused tissue, all indicators of tissue ischemia or cell necrosis, showed a good preservation of the organ for up to 5 hr. Carbachol was able to induce contractions of the prefilled bladder with a complete draining of the bladder throughout the whole perfusion period. Conclusions We could demonstrate that this perfusion system was able to preserve the pig bladder in a functional condition, appropriate for the study of physiological questions. Neurourol. Urodynam. 26:1024–1029, 2007. © 2007 Wiley-Liss, Inc.

OBJECTIVES To investigate whether the responses of the pig bladder to isoprenaline (a nonselective β-adrenoceptor agonist) are influenced by the presence of an intact urothelium and whether any influence might be attributed to the release of nitric oxide (NO), since stimulation of β-adrenoceptors induces a direct relaxation of detrusor smooth muscle and β-adrenoceptors are also present on the urothelium. MATERIAL AND METHODS Paired (in the presence or absence of urothelium) longitudinal strips of pig bladder dome were set up in tissue baths and the developed tension recorded. Relaxation responses to isoprenaline were examined after pre-contraction with carbachol. The inhibitory effects of isoprenaline were examined by comparing responses to carbachol in the absence and presence of isoprenaline. To examine a possible role for NO, similar experiments were performed in the presence of the NO synthase inhibitor NG-nitro-l-arginine (L-NNA). RESULTS In the presence of the urothelium, both the potency (pEC50) and the maximum contractile responses to carbachol were depressed. In relaxation experiments, isoprenaline relaxed carbachol pre-contracted tissues by ≈75%, and the potency and maximum relaxation were similar in the absence and presence of the urothelium. In the inhibition experiments, the presence of isoprenaline caused rightward parallel shifts of the concentration-response curves to carbachol, but isoprenaline did not influence the maximum contractions. In the presence of the urothelium there was a greater shift with 0.1 µm isoprenaline than in denuded tissues. Incubation with L-NNA did not affect the influence of the urothelium on responses to isoprenaline in any experimental group. CONCLUSIONS The relaxation responses of the bladder to isoprenaline do not appear to involve the urothelium or NO release in vitro. However, contractile responses to carbachol were inhibited in the presence of an intact urothelium, and this might reflect the release of an inhibitory factor other than NO.

Flavonoids comprise a large group of natural polyphenolic compounds, which possess a wide spectrum of physiological and pharmacological effects. Recently, the flavonoid galangin was found to modulate smooth muscle contractility. The aim of the present study was to investigate the mechanism of actions of galangin on pig bladder smooth muscle and to characterize its potential as an alternative inhibitor of bladder smooth muscle contraction.Strips of pig detrusor muscle were mounted in separate 6-ml organ baths containing Krebs solution. The contractile response to carbachol (10(-8)-10(-4)M), potassium (2x10(-2)-10(-1)M), and electrical field stimulation-EFS (2-32 Hz) were determined before and after the addition of galangin (3x10(-5)M). The contractile responses to carbachol in calcium-free Krebs' solution plus EGTA and L-type channel blocker were determined in the absence and presence of the flavonoid. Furthermore, the effect of galangin was also evaluated after the administration in the bath of a number of antagonists/inhibitors including a combination of propranolol, phentolamine, capsazepine, and verapamil. Student's t-test and one factor ANOVA were used to determine the statistical significance of the effects.Galangin inhibited the maximal contractile response to carbachol and potassium by 57.41% (P0.01) and 33.52% (P0.05), respectively. The maximum force of the carbachol-evoked contractions in calcium-free solution after incubation with galangin was 32% of the maximum initial force (Emax.initial: 5.8387+/-0.72 mN, Emax.Galangin: 1.9157+/-0.30 mN, P0.01). The maximal contractile responses to EFS at 2, 4, 8, 16, and 32 Hz were reduced, compared to control, by 91.61% (P0.01), 79.46% (P0.01), 70.54% (P0.01), 61.10% (P0.01), and 9.8% (P0.05), respectively. The inhibitory effect of galangin was unaffected by a combination of propranolol, phentolamine, and capsazepine (P0.05). However, when verapamil was added to the medium, the inhibitory effects of galangin were partially blocked.Galangin, at high concentrations, exerts an inhibitory effect on pig bladder smooth muscle contractility through the inhibition of calcium influx and the modulation of intracellular calcium movement. Furthermore, we have demonstrated that the inhibitory effect of galangin involves, at least in part, L-type calcium channels pathways.

Purpose: We determined whether the nature of any protective barrier in the bladder is composed of a secreted mucous gel layer. Materials and Methods: We collected 24-hour urine samples for analysis from 8 healthy 22 to 49-year-old volunteers and 5, 19 to 59-year-old patients treated with bladder reconstruction, in addition to scrapings from 100 freshly slaughtered pig bladders. Samples were subjected to homogenization, dialysis, freeze-drying, papain digestion, gel chromatography, equilibrium density gradient centrifugation, periodic acid-Schiff assay and amino acid analysis. Normal human bladder, pig bladder, normal ileum and transposed intestinal segments were studied for the presence of a mucous layer using a new method of histological analysis. Results: Mucin content in normal urine is 2.7 mg/24 hours, meaning that less than 0.6% of nondialyzable material in normal urine is mucin. The mucin content of urine from reconstructed bladders amounted to 86 mg/24 hours (5.2% of nondialyzable material). We observed that glycosaminoglycans accounted for 41% of the peak total elution volume of PAS positive material in normal urine. Mucin estimation in urine can be grossly overestimated if contaminating glycoconjugates are not removed. Biochemical analysis of material scraped off the pig bladder surface demonstrated that the maximum thickness of a continuous layer that could be achieved was 13.6 μm. While we could visualize an obvious mucous layer on control ileal samples and biopsies of transposed ileal segments from patients with bladder reconstruction, we were unable to note a distinct, measurable mucous layer lining the bladder surface in humans or pigs. Conclusions: Mucin levels in normal human and pig urine would be enough for slow turnover of a thin barrier but the large increase in mucin in the urine of patients with transposed intestinal segments demonstrates that any layer in normal bladder is much different than that lining the transposed intestinal segment. The most likely constituents of this barrier are membrane bound rather than secreted mucins along with the proteoglycan components of the glycocalix.

Objectives. To test the capability of the flavonoid galangin to protect pig urinary bladder from damage due to a period of repetitive field stimulation as well as a period of anoxia/glucopenia and reperfusion. Methods. Smooth muscle strips of the pig bladder were mounted for tension recording in small organ baths and the strips underwent either 1.5 hours of repetitive field stimulation at 32 Hz for 15 seconds every 5 minutes or under anoxia/glucopenia and reperfusion conditions. Galangin, at different concentrations, was added to the reperfusion Krebs solution to check the effect of this flavonoid compared with untreated strips under the same conditions. A group of experiments was performed to examine its possible underlying mechanisms. Results. Repetitive field stimulation for 1.5 hours caused a progressive decrease in the maximal contractile response to electrical field stimulation (34% decrease). Galangin (10 7 M) partially prevented the progressive decrease in the contractile response. This effect was significantly reduced when verapamil was added to the solution. Galangin significantly improved the response of strips to electrical field stimulation under anoxia/glucopenia and reperfusion conditions compared with untreated tissues. Conclusions. Galangin has a protective effect on bladder contractility by an action that at least, in part, depends on L-type calcium channels. Furthermore, galangin protects detrusor nerves against the anoxia/ glucopenic and reperfusion damage. UROLOGY 66: 1327‐1331, 2005. © 2005 Elsevier Inc.

Stimulating muscarinic receptors in pig bladder urothelium causes the release of a diffusable factor that inhibits contractions of the underlying detrusor muscle. We investigated whether the contractions of human detrusor strips elicited by the muscarinic agonist carbachol, electrical field stimulation, KCl or the neurokinin receptor agonist neurokinin A are affected by the urothelium.Paired intact and urothelium denuded muscle strips were placed in modified gassed Tyrode's solution at 37C. Cumulative concentration-response curves to carbachol or KCl were constructed. In other tissues the strips were stimulated electrically (1 to 40 Hz) with trains of square wave pulses 20 seconds in duration at 5-minute intervals.Cholinergic contractions evoked by electrical field stimulation at 10 and 30 Hz or by carbachol were significantly inhibited in the presence of an intact urothelium. Contractions elicited by KCl and by 10 microM neurokinin A were not modified by the urothelium. The urothelium mediated inhibition of contractions induced by carbachol was not affected by 300 microM L-NG-nitroarginine, 1 microM ODQ (1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one), 1 microM propranolol or 5 microM indomethacin.Muscarinic agonists stimulate the release of an inhibitory factor from the human urothelium. The factor is distinct from nitric oxide and it persists in the presence beta-adrenoceptor blockade or cyclooxygenase inhibition.

Intravesical electromotive administration of local anesthetics is clinically successful but electrochemistry, cost and effectiveness limit the choice of drugs to diluted lidocaine HCl 4% mixed with epinephrine. These studies address the stability of lidocaine and epinephrine both over time and when exposed to electric current, i.e. transport rates with passive diffusion and electromotive administration. The drug mixture used was 50 ml lidocaine 4%, 50 ml H2O and 1 ml epinephrine 1/1000. For stability, the solution was placed either in bowls for 7 days or in a two chamber cell with the donor compartment (drugs) separated from the receptor compartment (NaCl solution) by a viable pig bladder wall. This was subjected to 30 mA for 45 min. Stability was measured with mass spectrometry. The cell was also used to determine transport rates with passive diffusion and currents of 20 mA and 30 mA, over 20, 30 and 45 min. Drug measurements in both compartments and bladder were made with HPLC. Lidocaine remained stable throughout the 7 days, epinephrine on day 1 only and both drugs were stable with 30 mA for 45 min. Comparing 20 mA and 30 mA with passive diffusion, there were significant differences in 6/6 donor compartment lidocaine levels, 4/6 receptor compartment levels and 6/6 bladder tissue levels and also in 6/6 epinephrine donor levels and 6/6 tissue levels. The combination lidocaine and epinephrine remains stable for 1 day and when exposed to 30 mA for 45 min. Electric current accelerates the transport of lidocaine and epinephrine.

In the dome of the bladder of the pig muscarinic receptor stimulation has been shown to release a factor from the urothelium that exerts an inhibitory effect on the underlying smooth muscle. We examined whether the urothelium in the trigone of the bladder also releases this factor, identified which receptors stimulate its release and investigated possible cross-talk among these receptor systems in the trigone.Paired longitudinal strips of pig bladder were isolated, the urothelium was removed from 1 strip per pair and tissues were set up in gassed Krebs solution at 37C. Cumulative concentration-response curves to carbachol, phenylephrine or histamine were constructed. In some tissues a second phenylephrine curve was constructed in the presence of 1 microM. carbachol or 1 microM. histamine. In a further group of tissues the second phenylephrine curve was constructed in the presence of 1 microM. carbachol and 1 microM. atropine or 1 microM. histamine and 100 nM. mepyramine.In the presence of an intact urothelium contractile responses to carbachol and histamine but not to phenylephrine were depressed. In the presence of 1 microM. carbachol or 1 microM. histamine the responses of intact urothelium strips to phenylephrine were significantly depressed. This effect was absent in the presence of atropine and mepyramine, respectively.Carbachol and histamine induce the release of a urothelium derived inhibitory factor in the bladder trigone. The factor appears to mediate cross-talk between these systems and the alpha-adrenoceptor system in this region of the bladder.

In the bladder body M2 muscarinic receptors predominate but a smaller population of M3 receptors mediates direct detrusor contraction. M2 receptors have an indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body. We investigated whether a similar mechanism also exists in the bladder base.Experiments were performed on pig detrusor muscle. In receptor binding studies using l-quinuclidinyl [phenyl-4-(3)H] benzilate ([(3)H]QNB) (NEN Life Science Products, Inc., Boston, Massachusetts) displacement experiments were performed with subtype selective antagonists to determine the M2-to-M3 receptor ratio. In functional studies the affinity of these antagonists against carbachol induced contractions was calculated in normal tissues and in tissues after cyclic adenosine monophosphate elevation by pre-contraction with KCl and relaxation with isoprenaline, and/or selective M3 inactivation by incubation with 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) mustard (Sigma Chemical Co., St. Louis, Missouri) in the presence of methoctramine (Sigma Chemical Co.) to protect M2 receptors.In saturation binding studies receptor density was 130.5 of fmol./mg. protein and the dissociation constant for [(3)H]QNB was 0.27 nM. Displacement of [(3)H]QNB by the M3 selective antagonist 4-DAMP and the M2 antagonist methoctramine indicated an M2-to-M3 ratio of about 3:1. In functional studies 4-DAMP and methoctramine caused competitive antagonism of responses with affinity values of 9.5 and 6.3 in normal tissues, and 9.3 and 6.1, respectively, in cyclic adenosine monophosphate elevated tissues, suggesting the involvement of M3 receptors only. In tissues in which M3 receptors were inactivated and cyclic adenosine monophosphate levels were elevated the affinity of 4-DAMP was significantly reduced to 8.5 but that of methoctramine was significantly increased to 6.5, suggesting the involvement of M2 receptors.The M3 subtype appears to mediate contraction of the normal and cyclic adenosine monophosphate elevated tissues of the bladder base. Involvement of M2 receptors was only noted after selective M3 inactivation and cyclic adenosine monophosphate elevation.

ObjectivesThis article summarizes evidence supporting the conclusion that the spontaneous rises in pressure that occur in the overactive bladder, particularly in detrusor instability (DI), regardless of etiology, are myogenic. MethodsThe evidence quoted has been obtained by several groups of investigators and includes electrophysiologic studies of detrusor myocytes, tension studies of strips of smooth muscle, in vivo experiments on animal models, and light and electron microscopic studies of the structure of the bladder wall. Results.The results of these studies demonstrate changes in the properties, structure, and innervation of the detrusor. These changes are consistent with the hypothesis that partial denervation of the detrusor may be responsible for altering the properties of the smooth muscle, leading to increased excitability and increased ability of activity to spread between cells, resulting in coordinated myogenic contractions of the whole detrusor. Conclusions.lt is suggested that alterations in the properties of the detrusor myocytes are a necessary prerequisite for the production of the unstable pressure rises seen in DI of any origin.

Aims Alterations in properties of the bladder with maturation are relevant physiologically and pathophysiologically. The aim of this study was to investigate alterations in bladder properties with maturation in juvenile vs. adult pig, focussing on differences between layers of the bladder wall (mucosa vs. detrusor) and the presence and functional contribution of interstitial cells (ICs). Methods Basal and cholinergic-induced phasic contractions (PCs) in mucosal and denuded-detrusor strips from juvenile and adult pigs were assessed. Expression of c-kit, a marker of ICs, was investigated in the mucosa and the detrusor layers of the pig bladder. The functional role of ICs in mediating PCs was examined using imatinib. Results Mucosal strips from juvenile and adult pig bladders demonstrated basal PCs whilst denuded-detrusor strips did not. PCs of mucosal strips from juvenile pigs were significantly greater than those from adult bladders. Immunoreactivity for c-kit was detected in mucosa and detrusor layers of pig bladder. Histological studies demonstrated a distinct layer of smooth muscle between the urothelium and bladder detrusor, termed the muscularis mucosa. Imatinib was only effective in inhibiting PCs in mucosal strips from juvenile pigs. Imatinib inhibited the carbachol-induced PCs of both juvenile and adult denuded-detrusor strips, although strips from juvenile bladders demonstrated a trend towards being more sensitive to this inhibition. Conclusions We confirm the presence of c-kit positive ICs in pig urinary bladder. The enhanced PCs of mucosal strips from juvenile animals could be due to altered properties of ICs or the muscularis mucosa in the bladders of these animals.

Hypothesis / aims of study: Urothelial ATP release is thought to play an important role in bladder afferent signaling via activation of purinergic receptors on suburothelial afferent nerves. Stretch of the bladder mucosa is a well documented stimulus for ATP release in several species, including the pig [1]. In addition, in mouse bladder, capsaicin is also an effective stimulus for ATP release, acting via stimulation of vanilloid (TRPV1) receptors [2]. While acid is an agonist at the TRPV1 receptor, specialized acid sensing ion channels (ASICs) are also present in several organ systems [3]. Our aim was to characterize the ATP release from pig bladder mucosa in response to stretch, acid and capsaicin.