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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

Epigallocatechin gallate (EGCG) and tannic acid (TA) are known to increase the aqueous solubility and cellular uptake of the hydrophobic drugs docetaxel, paclitaxel, amphotericin B, and curcumin. In this study the practical application of gallate-based solubilization phenomena for the uptake of these drugs into dermal and bladder tissue and of lidocaine for wound healing application was studied. The penetration of all these drugs into pig skin or docetaxel into pig bladder using EGCG or TA formulations was measured. Overall, EGCG and TA particulate or propylene glycol paste formulations of drugs allowed for greatly increased levels of drug uptake into skin as compared to control formulations. EGCG/propylene glycol pastes allowed for rapid lidocaine uptake into skin. EGCG and TA formulations of docetaxel allowed for approximately 10 fold increases in bladder tissue uptake of docetaxel over tween based solutions. Morphologically, both EGCG and TA caused a mild, dose dependent exfoliation of the bladder wall. Both EGCG and TA formed injectable viscous pastes with propylene glycol which solidified in water and degraded and released lidocaine over 2-35 days. These data support the use of EGCG and TA based formulations of certain drugs for improved dermal, bladder and wound applications.

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...

Accurate measurement of bladder volume is an important tool for evaluating bladder function. In this study, we propose a wearable bladder scanner system that can continuously measure bladder volume in daily life for urinary patients who need urodynamic studies. The system consisted of a 2-D array, which included integrated forward-looking piezoelectric transducers with thin substrates. This study aims to estimate the volume of the bladder using a small number of piezoelectric transducers. A least-squares method was implemented to optimize an ellipsoid in a quadratic surface equation for bladder volume estimation. Ex-vivo experiments of a pig bladder were conducted to validate the proposed system. This work presents the potential of the approach for wearable bladder monitoring, which has similar measurement accuracy compared to the commercial bladder imaging system. The wearable bladder scanner can be improved further as electronic voiding diaries by adding a few more features to the current function.

OBJECTIVE: In a concept of accommodation of detrusor pressure to volume as an autonomous potency of the bladder, a crucial physiological biomechanical role has been attributed to spontaneous contraction activity. This concept is experimentally investigated on pig bladder in vitro.METHODS: Slowly emptying of not-stimulated pig bladders via a flow resistor has been recorded and the effect of spontaneous contractions on the tonic pressure during emptying by expulsion has been studied.RESULTS: The expulsed volume can be separated in a reduction of elastic volume and of rest volume. Tonic pressure is determined by the elastic volume in combination with elastic compliance. In an accommodated state completely transient superimposed pressure waves affect rest volume not elastic volume. Accommodation of tonic detrusor pressure to bladder volume is based on equilibration between passive elongations and active transient contractions distributed in bladder wall.CONCLUSION: Maintenance of a tonic accommodated detrusor pressure to a constant or slowly varying volume, obtained by a process of equilibration between passive elongations and active contractions, can be understood as an autonomous potency of a bladder. The earlier presented concept of active accommodation has been validated by the experiments. The pressure-volume relation of the bladder is fundamentally revised. Total volume V can be virtually separated in an elastic volume VE and a plastic or rest volume VR. Both parts change with V and in changing ratio. Tonic pressure marks a border between VE and VR.

Metabolic Syndrome (MetS) has detrimental effects on the bladder, including detrusor underactivity. The progression and mechanism of disease are poorly understood. A swine model for diabetic bladder dysfunction (DBD) was established because of the pig's human-sized bladder and its ability to develop MetS by dietary modification alone. The hypothesis of this study is that this swine model will demonstrate oxidative stress associated with MetS, which contributes to both bladder fibrosis and detrusor underactivity (DU). Ossabaw pigs underwent dietary modification consisting of a hypercaloric, atherogenic diet for 10 mo to induce MetS, and were compared with a group of control (lean) pigs. Urodynamic studies were performed in both groups to confirm DU. Thiobarbituric acid reactive substances (TBARS) detected in the urine were used to measure oxidative stress activity in the urinary tract, and urinary IL17a was used to detect profibrotic activity. MetS was confirmed by assessing body weight, blood pressure, glucose tolerance, total cholesterol, and triglycerides. The MetS group exhibited an increase in the relative levels of urinary TBARS and IL17a. Bladder pressures at capacity were lower in the MetS group, suggesting DU. Histologic analysis of a cohort of control (lean) and MetS pigs revealed that as compared with the control pigs, the MetS pigs had significantly more collagen in the muscularis layer, but not in the submucosa or mucosa layer. In conclusion, the Ossabaw pig model for diet-induced MetS is associated with oxidative stress and profibrotic activity in the bladder, which results in DU. This has previously been shown in mice and rats, but never in pigs. This novel model will better represent human MetS and DBD because the mechanism and size of the pig bladder more closely resemble that of a human, resulting in a more valid model and facilitating further study into the signaling mechanisms responsible for this impairment.

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.

Bladder cancer, with the highest recurrence rate in all malignancy, is a common urologic cancer that arises on the bladder mucosa. Currently, tumor resection followed by intravesical chemotherapy is the primary treatment of bladder cancer, which has limited effectiveness ascribe to short dwell-time of intravesical drugs in bladder. Therefore, there is a need to develop mucoadhesive and sustained drug delivery systems to increase drug residence time for intravesical chemotherapy. In this study, poly(amidoamine) (PAMAM) dendrimers were modified onto the surface of mesoporous silica nanoparticles (MSNPs) through a layer-by-layer grafting method. A series of PAMAM-modified MSNPs were prepared and compared for their mucoadhesive capabilities on pig bladder wall and controlled drug release properties. Results demonstrated an increase in the mucoadhesive capacity of PAMAM-modified MSNPs upon an increase in the number of PAMAM amino groups, and the maximum nanoparticle mucoadhesivity was observed after two-generation PAMAM were grafted on the surface of MSNPs. An antineoplastic, doxorubicin, was encapsulated in the mesopores of PAMAM-modified MSNPs, and the drug-loaded nanoparticles can provide a sustained drug release triggered by acidic pH. The present study demonstrates that the mucoadhesive and drug release properties of MSNPs can be controlled by the layer number of PAMAM dendrimers on the nanoparticle surface, holding significant potential for the development of mucoadhesive drug delivery systems for bladder cancer therapy.

Background: The interleukin-1-receptor antagonist IL1RA (encoded by the IL1RN gene) is a potent competitive antagonist to interleukin-1 (IL1) and thereby is mainly involved in the regulation of inflammation. Previous data indicated a role of IL1RA in muscle-invasive urothelial carcinoma of the bladder (UCB) as well as an IL1-dependent decrease in tissue barrier function, potentially contributing to cancer cell invasion. Objective: Based on these observations, here we investigated the potential roles of IL1RA, IL1A, and IL1B in bladder cancer cell invasion in vitro. Methods: Cell culture, real-time impedance sensing, invasion assays (Boyden chamber, pig bladder model), qPCR, Western blot, ELISA, gene overexpression. Results: We observed a loss of IL1RA expression in invasive, high-grade bladder cancer cell lines T24, UMUC-3, and HT1197 while IL1RA expression was readily detectable in the immortalized UROtsa cells, the non-invasive bladder cancer cell line RT4, and in benign patient urothelium. Thus, we modified the invasive human bladder cancer cell line T24 to ectopically express IL1RA, and measured changes in cell migration/invasion using the xCELLigence Real-Time-Cell-Analysis (RTCA) system and the Boyden chamber assay. The real-time observation data showed a significant decrease of cell migration and invasion in T24 cells overexpressing IL1RA (T24-IL1RA), compared to cells harboring an empty vector (T24-EV). Concurrently, tumor cytokines, e.g., IL1B, attenuated the vascular endothelial barrier, which resulted in a reduction of the Cell Index (CI), an impedance-based dimensionless unit. This reduction could be reverted by the simultaneous incubation with IL1RA. Moreover, we used an ex vivo porcine organ culture system to evaluate cell invasion capacity and showed that T24-IL1RA cells showed significantly less invasive capacity compared to parental T24 cells or T24-EV. Conclusions: Taken together, our results indicate an inverse correlation between IL1RA expression and tumor cell invasive capacity and migration, suggesting that IL1RA plays a role in bladder carcinogenesis, while the exact mechanisms by which IL1RA influences tumor cells migration/invasion remain to be clarified in future studies. Furthermore, we confirmed that real-time impedance sensing and the porcine ex vivo organ culture methods are powerful tools to discover differences in cancer cell migration and invasion.

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

An implantable sensor system has been developed to explore the physiological behaviour of the bladder wall. It consists of a 3-axis digital accelerometer and a pressure sensor on the upper side of a flexible polyimide printed circuit board. On the bottom side, two metallic hooks prevent migration of the device in the bladder wall while simultaneously acting as electrodes to record the electrical activity of the detrusor muscle. The full system is inserted at the interface between the detrusor and the mucosa, preventing exposure of the transducer to the urine. This ensures protection both for inflammation of the inner bladder, and from salts deposition on the device. Three packaging layers have been applied to ensure biocompatibility of the device: Parylene-C, medical epoxy and PDMS. The device was first tested in-vitro for water diffusion. Then, the feasibility of submucosal implant was demonstrated in a cadaver pig bladder, and in-vivo recordings were successfully acquired in a rat. ispartof: pages:0-0 ispartof: Proceedings vol:1 issue:4 pages:0-0 ispartof: Eurosensors location:Paris date:3 Sep - 6 Sep 2017 status: published

More than 90 % of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5 μM BaP for up to 24 h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70 μM in cells exposed to 0.5 μM BaP and from 29.9 to 406.64 μM in cells exposed to 10 μM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.

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.

BACKGROUND AND PURPOSE\ud \ud As adenosine 5'-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck.\ud \ud EXPERIMENTAL APPROACH\ud \ud Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37 degrees C and gassed with 95% O(2) and 5% CO2 for isometric force recordings.\ud \ud KEY RESULTS\ud \ud EFS, in the presence of atropine, guanethidine and N(G)-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5'-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5'-triphosphate, uridine 5'-diphosphate and alpha,beta-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y(1) and P1 receptor blockers 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist's potency was: 5'-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N(6)-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A(2A) antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na(+) channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X(1) and P2X(3) receptors.\ud \ud CONCLUSIONS AND IMPLICATIONS\ud \ud ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A(2A) receptors after breakdown to adenosine, and P2Y(1) receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency.

BACKGROUND AND PURPOSE 5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck. EXPERIMENTAL APPROACH Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS). KEY RESULTS After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (+/-)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > alpha-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT(7) receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT(1A) and 5-HT(1A/1B) receptor antagonists respectively. Inhibitors of 5-HT(1B/1D), 5-HT(2), 5-HT(2B/2C), 5-HT(3), 5-HT(4), 5-HT(5A) and 5-HT(6) receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, alpha-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca(2+)-activated K(+) and ATP-dependent K(+) channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na(+)-, Ca(2+)- and voltage-gated K(+) (K(v))-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol. CONCLUSIONS AND IMPLICATIONS 5-HT relaxed the pig urinary bladder neck through muscle 5-HT(7) receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT(1A) receptors and K(v) channels modulated 5-HT-induced relaxations whereas postjunctional K(+) channels were not involved in such responses. 5-HT(7) receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.

Background and purpose: The bladder urothelium is now known to have active properties. Our aim was to investigate the contractile properties of the urinary mucosa in response to the tachykinin neurokinin A (NKA) and carbachol. Experimental approach: Discrete concentration–response curves for carbachol and NKA were obtained in matched strips of porcine detrusor, mucosa and intact bladder, suspended in organ baths. The effects of inhibitors and tachykinin receptor antagonists were studied on NKA-mediated contractions in mucosal strips. Intact sections of bladder and experimental strips were processed for histology and immunohistochemistry. Key results: All types of strips contracted to both carbachol and NKA. Mucosal responses to NKA (pD2 7.2) were higher than those in intact strips and were inhibited by the NK2 receptor antagonist SR48968 (pKB 9.85) but not the NK1 receptor antagonist SR140333, tetrodotoxin or indomethacin. Immunostaining for smooth muscle actin and vimentin occurred under the urothelium and on blood vessels. Desmin immunostaining and histological studies showed only sparse smooth muscle to be present in the mucosal strips. Removal of smooth muscle remnants from mucosal strips did not alter the responses to NKA. Conclusions and implications: This study has shown both functional and histological evidence for contractile properties of the mucosa, distinct from the detrusor. Mucosal contractions to NKA appear to be directly mediated via NK2 receptors. The main cell type mediating mucosal contractions is suggested to be suburothelial myofibroblasts. Mucosal contractions may be important in vivo for matching the luminal surface area to bladder volume. British Journal of Pharmacology (2008) 153, 1465–1473; doi:10.1038/bjp.2008.29; published online 11 February 2008

Background and Purpose: ATP-sensitive K+ channels (KATP) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4-methyl-N-(2,2,2-trichloro-1-(3-pyridin-3-ylthioureido)ethyl)benzamide (A-251179) as a KATP channel opener. Experimental Approach: Glyburide-sensitive membrane potential, patch clamp and tension assays were employed to study the effect of A-251179 in vitro. The in vivo efficacy of A-251179 was characterized by suppression of spontaneous contractions in obstructed rat bladder and by measuring urodynamic function of urethane-anesthetized rat models. Key Results: A-251179 was about 4-fold more selective in activating SUR2B-Kir6.2 derived KATP channels compared to those derived from SUR2A-Kir6.2. In pig bladder smooth muscle strips, A-251179 suppressed spontaneous contractions, about 27- and 71-fold more potently compared to suppression of contractions evoked by low-frequency electrical stimulation and carbachol, respectively. In vivo, A-251179 suppressed spontaneous non-voiding bladder contractions from partial outlet-obstructed rats. Interestingly, in the neurogenic model where isovolumetric contractions were measured by continuous transvesical cystometry, A-251179 at a dose of 0.3 μmol kg-1, but not higher, was found to increase bladder capacity without affecting either the voiding efficiency or changes in mean arterial blood pressure. Conclusions and Implications: The thioureabenzamide analog, A-251179 is a potent novel KATP channel opener with selectivity for SUR2B/Kir6.2 containing KATP channels relative to pinacidil. The pharmacological profile of A-251179 is to increase bladder capacity and to prolong the time between voids without affecting voiding efficiency and represents an interesting characteristic to be explored for further investigations of KATP channel openers for the treatment of overactive bladder. British Journal of Pharmacology (2007) 151, 467–475; doi:10.1038/sj.bjp.0707249

Recent advances in microelectronics and wireless transmission technology have led to the development of various implantable sensors for real-time monitoring of bladder conditions. Although various sensing approaches for monitoring bladder conditions were reported, most such sensors have remained at the laboratory stage due to the existence of vital drawbacks. In the present study, we explored a new concept for monitoring the bladder capacity on the basis of potentiometric principles. A prototype of a potentiometer module was designed and fabricated and integrated with a commercial wireless transmission module and power unit. A series of in vitro pig bladder experiments was conducted to determine the best design parameters for implementing the prototype potentiometric device and to prove its feasibility. We successfully implemented the potentiometric module in a pig bladder model in vitro, and the error of the accuracy of bladder volume detection was &lt, ±3%. Although the proposed potentiometric device was built using a commercial wireless module, the design principles and animal experience gathered from this research can serve as a basis for developing new implantable bladder sensors in the future.

Item does not contain fulltext BACKGROUND AIMS: On review of the use of stem cells in the literature, promissory outcomes for functional organ recovery in many subspecialties in medicine underscore its therapeutic potential. The application of stem cells through the use of a needle can result in additional scar formation, which is undesired for delicate organs. The present work describes the use of a needle-less stem cell injector with the Immediate Drop on Demand Technology (I-DOT) for cell injection in vitro. METHODS: Mesenchymal stromal cells from human bone marrow were labeled with ethynyl-deoxyuridine (EdU) for 2 days and then were re-suspended. With the use of I-DOT, the cells were applied to type 1 collagen matrices or pig bladder tissue specimens with or without mucosa at different levels of energy. The collagen matrices were analyzed after 4 h and 5 days; bladder tissue specimens were analyzed 4 h after cell implantation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) assay was performed immediately after cell application to the collagen matrices. Histological analysis with the use of frozen sections and immunofluorescence was used to localize EdU-labeled cells. RESULTS: A considerable number of cells were detected by use of the MTT assay for collagen matrices. In the collagen matrix, the mean measured depth immediately after application ranged between 210 mum and 489 mum, 220 mum and 270 mum for entire bladder specimens, and 230 mum and 370 mum for bladder without mucosa. Cells survived for up to 5 days in the collagen matrix in both bladder specimens. CONCLUSIONS: Cells can survive during I-DOT application, which suggests that the I-DOT device may be a potentially suitable technology for needle-less cell application onto tissues.

Background and purpose: As pituitary adenylate cyclase-activating polypeptide 38 (PACAP 38)- and vasoactive intestinal peptide (VIP) are widely distributed in the urinary tract, the current study investigated the receptors and mechanisms involved in relaxations induced by these peptides in the pig bladder neck. Experimental approach: Urothelium-denuded strips were suspended in organ baths for isometric force recordings and the relaxations to VIP and PACAP analogues were investigated. Key results: VIP, PACAP 38, PACAP 27 and [Ala11,22,28]-VIP produced similar relaxations. Inhibition of neuronal voltage-gated Ca2+ channels reduced relaxations to PACAP 38 and increased those induced by VIP. Blockade of capsaicin-sensitive primary afferents (CSPA), nitric oxide (NO)-synthase or guanylate cyclase reduced the PACAP 38 relaxations but failed to modify the VIP responses. Inhibition of VIP/PACAP receptors and of voltage-gated K+ channels reduced PACAP 38 and VIP relaxations, which were not modified by the K+ channel blockers iberiotoxin, charybdotoxin, apamin or glibenclamide. The phosphodiesterase 4 inhibitor rolipram and the adenylate cyclase activator forskolin produced potent relaxations. Blockade of protein kinase A (PKA) reduced PACAP 38- and VIP-induced relaxations. Conclusions and implications: PACAP 38 and VIP relax the pig urinary bladder neck through muscle VPAC2 receptors linked to the cAMP-PKA pathway and involve activation of voltage-gated K+ channels. Facilitatory PAC1 receptors located at CSPA and coupled to NO release, and inhibitory VPAC receptors at motor endings are also involved in the relaxations to PACAP 38 and VIP, respectively. VIP/PACAP receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency. British Journal of Pharmacology (2006) 149, 100–109. doi:10.1038/sj.bjp.0706832

The electrophysiological properties of detrusor smooth muscles have been studied almost exclusively in small mammals and the relevance of the information to the human bladder has been questioned. In the present study, electrical properties of detrusor smooth muscles of the pig and human were investigated using intracellular recording techniques. Bladder smooth muscles of the pig and human exhibited nifedipine (10 μM)-sensitive spontaneous action potentials, and their frequency was highly sensitive to membrane polarization. During bursts of action potentials, each action potential was followed by a fast after-hyperpolarization (fast AHP). Charybdotoxin (CTX, 50 nM) increased the amplitude and duration of action potentials but failed to inhibit the fast AHPs, while apamin (0.1 μM) blocked the fast AHPs and induced action potential complexes, which were followed by slow AHPs. 4-Aminopyridine (4-AP, 1 mM) suppressed the slow AHP and increased action potential frequency. In the human bladder, transmural stimuli initiated inhibitory junction potential-like hyperpolarizations, which were followed by action potential discharges. The hyperpolarizations were blocked by atropine (1 μM) and by apamin (0.1 μM) but not by CTX (50 nM). In the pig bladder, transmural stimuli evoked excitatory junction potentials (EJPs), which triggered action potentials. After desensitizing P2x receptors with α,β methylene-ATP (10 μM), nerve-evoked responses were similar to those of human bladder. These results indicate that detrusor smooth muscles of the pig share many features of electrical properties with those of the human. In addition to large conductance (BK) and small conductance (SK) Ca2+-activated K+ channels, voltage-dependent K+ (VK) channels may play an important role in the regulation of electrical activity of detrusor smooth muscles. British Journal of Pharmacology (2003) 140, 146–158. doi:10.1038/sj.bjp.0705319

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.

A hallmark for unstable bladder contractions is hyperexcitability and changes in the nature of spontaneous phasic activity of the bladder smooth muscle. In this study, we have characterized the spontaneous activity of the urinary bladder smooth muscle from the pig, a widely used model for studying human bladder function. Our studies demonstrate that phasic activity of the pig detrusor is myogenic and is influenced by the presence of urothelium. Denuded strips exhibit robust spontaneous activity measured as mean area under the contraction curve (AUC=188.9+/-15.63 mNs) compared to intact strips (AUC=7.3+/-1.94 mNs). Spontaneous phasic activity, particularly the amplitude, is dependent on both calcium entry through voltage-dependent calcium channels and release from ryanodine receptors as shown by inhibition of spontaneous activity by nifedipine and ryanodine respectively. Inhibition of BK(Ca) channels by iberiotoxin (100 nM) resulted in an increase in contraction amplitude (89.1+/-20.4%) and frequency (92.5+/-31.0%). The SK(Ca) channel blocker apamin (100 nM) also increased contraction amplitude (69.1+/-24.3%) and frequency (53.5+/-13.6%) demonstrating that these mechanisms are critical to the regulation of phasic spontaneous activity. Inhibition of K(ATP) channels by glyburide (10 microM) did not significantly alter myogenic contractions (AUC=18.5+/-12.3%). However, K(ATP) channel openers (KCOs) showed an exquisite sensitivity for suppression of spontaneous myogenic activity. KCOs were generally 15 fold more potent in suppressing spontaneous activity compared to contractions evoked by electrical field-stimulation. These studies suggest that potassium channel modulation, particularly K(ATP) channels, may offer a unique mechanism for controlling spontaneous myogenic activity especially those associated with the hyperexcitability occurring in unstable bladders.

1 b-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of b3-adrenoceptors to relaxation of the pig urinary bladder. 2 Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [ 3 H]-dihydroalprenolol (DHA), a non-selective b-adrenoceptor antagonist was used as a specific radioligand to determine the presence of b-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the aAnity of antagonists. 3 In competition binding experiments, CGP20712A (b1-adrenoceptor selective) displaced [ 3 H]DHA from a single binding site with a low aAnity. In contrast, displacement data for ICI 118551 (b2-adrenoceptor antagonist) and SR59230A (b3-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of b3-adrenoceptors. 4 In functional studies, isoprenaline and salbutamol (b2-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC50 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (b3-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (b1-adrenoceptor antagonists) antagonised responses with a low aAnity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high aAnity (pKB=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one b-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pKB values for 3‐10 nM SR59230A being 8.6 and those for 30 nM‐1mM being 7.7. 5 These data suggest that b3-adrenoceptors are the predominant b-adrenoceptor subtype present in the pig bladder and that b-adrenoceptor mediated responses of this tissue are mediated via both the b2- and b3-adrenoceptor subtypes. British Journal of Pharmacology (2002) 135, 129‐134

Bladder wall mechanical properties are important indicators of bladder compliance. This study compares the effectiveness of the Kelvin-Voigt, Maxwell, and fractional Kelvin-Voigt rheological models in capturing the mechanical properties of normal (compliant) and aberrant (non-compliant) ex-vivo pig bladder walls. Bladders were filled to different volumes, varying their thickness and internal pressure, and excited by the acoustic radiation force. Pulse-echo ultrasound imaged anti-symmetrical Lamb waves traveling along the bladder wall. Two-dimensional Fourier analysis generated velocity-frequency dispersion curves for the Lamb wave propagation. Fitting these experimental curves against theoretical curves from the models yielded values for bladder elasticity and viscosity. Pressure, measured simultaneously, was used as a point of comparison with elasticity. Our findings indicate an increasing trend in both elasticity for the rheological models and recorded pressure with increasing volume. This rate was obse...

Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10–14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.

1. In urinary bladder, M(2)-muscarinic receptors predominate, but it is the smaller population of M(3)-receptors which mediate detrusor contraction. This study examines the M(2) : M(3) ratio and the role of M(2)-receptors in contraction of pig urinary bladder. 2. Competition experiments with [(3)H]-QNB determined the ratio of M(2) : M(3). In functional studies, affinity values (pK(B)) for 4-DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M(3)-inactivation (incubation with 40 nM 4-DAMP mustard in the presence of 1 microM methoctramine to protect M(2)-receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 microM) or forskolin (1 microM). 3. In competition binding, displacement of [(3)H]-QNB by 4-DAMP, darifenacin and methoctramine best fitted a two-site model suggesting a predominant (70 - 80%) population of M(2)-receptors. 4. On normal detrusor in vitro, 4-DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pK(B) values of 9.37+/-0.07 and 6.05+/-0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pK(B) value being 8.61+/-0.10. 5. In tissues where the M(3)-receptors had been inactivated and cyclic AMP levels elevated, 4-DAMP and darifenacin were less potent, with apparent pK(B) values of 8.72+/-0.08 and 6.74+/-0.07. In contrast, methoctramine was more potent, the apparent pK(B) value increasing significantly to 6.86+/-0.06. 6. se data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M(3)-receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M(2)-receptors in contraction can be observed following pharmacological manipulation of the receptor population.

The function of the bladder urothelium in modulating contractile responses of the underlying detrusor smooth muscle to muscarinic stimulation has been examined in the pig bladder. Saturation curves for [3H]-QNB binding demonstrated a greater muscarinic receptor density in the urothelium than in the detrusor smooth muscle. The presence of an intact urothelium on isolated bladder strips inhibited contractions induced by carbachol but not KCl. Contractions of a urothelium-denuded muscle strip were inhibited in the presence of a second bladder strip with an intact urothelium, but not if the second strip was denuded. The urothelium-induced inhibition of contractions was not prevented in the presence of L-NOARG, methylene blue, indomethacin, propranolol, suramin, TEA or apamin. The data suggest the presence of a diffusable, urothelium-derived inhibitory factor, which could not be identified but appears to be neither nitric oxide, a cyclo-oxygenase product, a catecholamine, adenosine, GABA nor an EDHF sensitive to apamin.

Flexible cystoscopy is frequently performed for recurrent bladder cancer surveillance, making it the most expensive cancer to treat over the patient's lifetime. An automated bladder surveillance system is being developed to robotically scan the bladder surface using an ultrathin and highly flexible endoscope. Such a system would allow cystoscopic procedures to be overseen by technical staff while urologists could review cystoscopic video postoperatively. In this paper, we demonstrate a method for reconstructing the surface of the whole bladder from endoscopic video using structure from motion. Video is acquired from a custom ultrathin and highly flexible endoscope that can retroflex to image the entire internal surface of the bladder. Selected frames are subsequently stitched into a mosaic and mapped to a reconstructed surface, creating a 3-D surface model of the bladder that can be expediently reviewed. Our software was tested on endoscopic video of an excised pig bladder. The resulting reconstruction possessed a projection error of 1.66 pixels on average and covered 99.6% of the bladder surface area.

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.

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10-14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.

Background: Vesicoureteral reflux (VUR) is a serious health problem leading to renal scarring in children. Current VUR detection involves traumatic x-ray imaging of kidneys following injection of contrast agent into bladder via invasive Foley catheter. We present an alternative non-invasive approach for detecting VUR by radiometric monitoring of kidney temperature while gently warming the bladder. Methods: We report the design and testing of: i) 915MHz square slot antenna array for heating bladder, ii) EMI-shielded log spiral microstrip receive antenna, iii) high-sensitivity 1.375GHz total power radiometer, iv) power modulation approach to increase urine temperature relative to overlying perfused tissues, and v) invivo porcine experiments characterizing bladder heating and radiometric temperature of aaline filled 30mL balloon "kidney" implanted 3-4cm deep in thorax and varied 2-6°C from core temperature. Results: SAR distributions are presented for two novel antennas designed to heat bladder and monitor deep kidney temperatures radiometrically. We demonstrate the ability to heat 180mL saline in in vivo porcine bladder to 40-44°C while maintaining overlying tissues

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.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+ -dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.

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.

The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 105 cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells on...

1. Water and isotope fluxes were measured by incubating urinary bladders of foetal pigs and sheep in vitro in the presence and absence of a concentration and osmotic gradient. The structure of the urinary bladder of foetal pigs under various conditions was studied by electron microscopy. Its ultrastructure was found to be closely similar to that of foetal sheep. 2. Antidiuretic hormone (ADH) (0-2 U. ml-1) enhanced the enlargement of intercellular spaces caused by dilute mucosal medium in pig bladders; prolactin (1 u. ml-1) prevented osmotic dilatation of the intercellular spaces. 3. The hydraulic conductivity, Lp, was estimated to be 0-5 X 10 (-7) cm.s-1atm-1 in sheep and pigs at about 100 days gestation; the ratio of osomotic to diffusional permeability, (LpRT/VW)/PD, in the presence and absence of ADH, was 2-1 and 1-6 respectively. These are similar to the values found in fish gills. 4. Prolactin reduced bulk flow of water to zero in seven out of eight bladders investigated. Incubation with ADH or vasotocin (55 mu. ml-1) in the presence of prolactin restored water flux to 22% and 45% of control values respectively. 5. There was no significant net flux of sodium from mucosa to serosa in pig bladder except in the presence of prolactin. No net flux of sodium occurred from mucosal to serosal side of pig or sheep bladders in the presence of an adverse electrochemical gradient, although in sheep the permeability ratio was significantly greater than one. 6. The diffusional flux ratio for water remained unity under all conditions; vasotocin increased unidirectional fluxes and prolactin reduced them. The flux ratios were unaffected by the direction of bulk fluid flow, probably because diffusion was rapid compared to flow: the ratio of diffusional flux to volume flow was between 11 and 18.

Strips of pig bladder have been maximally stimulatedin vitro at 37°C via electrodes placed in the muscle, in order, particularly, to measure the dependence of the resulting active force on the velocity of shortening and on length changes. The active isometric force and the passive viscoelastic force are approximately, but not precisely, additive. The active isometric force, like the steady (equilibrium) passive force, is a function of the extension of the strip above its rest length, which is increased after subjection to a high passive force. The steady passive force increases quasiexponentially with this extension, of which it is therefore a measure. The active isometric force Fiso increases approximately linearly with the extension until it approaches a maximum in the region where it and the steady passive force are comparable in size. The maximum is partly obscured by rest-length changes. The dependence of the active force F on the speed of shortening of the strip has been measured in a new way, with a correction for passive viscoelastic effects. For a given strip the ratio F/Fiso is, approximately, a function of the contraction velocity only. The function is similar to that of the classical Hill equation but not identical, possibly for geometrical reasons. The results imply that a velocity parameter v*, analogous to Hill’s parameter b, is approximately constant for each strip, independent of changes of length and rest length.

Strips of urinary bladder smooth muscle were subjected to a series of quick release measurements. Each measurement consisted of several releases and resets to the original length, made during one contraction. The complete length-force characteristic of series elasticity was quantified by estimating H, the amplitude of quick release necessary to reduce the active force to exactly zero, and Db, a measure for the deviation of the characteristic from a straight line. By measuring a series of contractions at increasing stretched strip lengths, the length dependence of these parameters was studied. It was found that H depends linearly on stretched strip length. On average H/length amounted to 0.04. Db decreased when strips were stretched, i.e. a straight line was more closely approximated. Both parameter dependencies support the concept of two separate elastic mechanisms, a linear true passive elasticity in series with a non-linear elasticity in the cross-bridges. For the latter, H amounts to 3.8% of the initial strip length.

Spontaneous activity in bladder muscle strips from man, pig and rabbit has been compared using an in vitro superfusion technique. Field stimulation was used to study nerve-mediated activity. Bladder muscle strips from all areas of the rabbit bladder displayed rhythmic spontaneous activity. Spontaneous activity was regularly present in strips from the trigone region in man and pig, but was present in only 18 and 19% respectively of strips from the dome of the bladder. Strength-duration curves in the presence of tetrodotoxin (10(-7) g/ml) were constructed. The 'chronaxie' of the muscle was found to be considerably shorter than that of other smooth muscles, ranging from 6.1 ms in the rabbit to 12.9 ms in man. Frequency-response curves were constructed using trains of stimuli. The responses were not antagonized by hexamethonium (10(-4) M), but were markedly inhibited by tetrodotoxin (10(-7) g/ml), indicating that the responses were mediated by excitation of post-ganglionic nerves. Physostigmine (10(-7)-5 X 10(-6) M) produced a dose-related increase in the contractile response to field stimulation in all three species. Atropine (10(-8)-10(-6) M) produced an inhibition of the contractile response, but the maximum degree of inhibition differed considerably between the species. In the rabbit, 58% of the control response was attained, whilst in the pig this was only 22%. Atropine completely abolished nerve-mediated contractions in human bladder muscle. Phentolmaine (10(-7)-2.5 X 10(-7) M) had no significant effect on the frequency-response curve in any of the three species, and did not depress the atropine-resistant component in rabbit and pig. It is concluded that nerve-mediated activity in human bladder muscle is exclusively cholinergic, in contrast to most other mammals studied in which there is a significant non-cholinergic component. The finding of a shorter chronaxie in bladder muscle than in other smooth muscles suggests important differences in its physiological properties that merit further investigation.

1. The effects of alpha,beta-methylene ATP (alpha,beta-MeATP) on membrane properties and excitatory junction potentials (EJPs) were examined in smooth muscle cells of the guinea-pig, rabbit and pig bladder. 2. Intracellular recording with microelectrodes was used to record membrane electrical activity from the guinea-pig bladder. ATP (10(-3) M) produced a rapid, large depolarization with a marked increase in spike frequency, while carbachol (10(-4) M) or acetylcholine (ACh; 10(-4) M) produced only a small or no depolarization with a smaller increase in spike frequency. alpha,beta-MeATP produced a similar response to that of ATP but at a much lower concentration (5 x 10(-6) M), and the response was transient even in the continuous presence of this agent. 3. Changes in the membrane potential and conductance elicited by alpha,beta-MeATP were also measured with the double sucrose-gap method. alpha,beta-MeATP (5 x 10(-6) M) depolarized the membrane and increased the membrane conductance in all three species, but both parameters returned to control values during continuous exposure to this agent. 4. Intracellular recording with microelectrodes showed that in the guinea-pig bladder treatment with alpha,beta-MeATP abolished the response to ATP, while the response to ACh was unchanged. 5. With the double sucrose-gap method, EJPs were elicited by transmural nerve stimulation of strips of the guinea-pig, rabbit and pig bladder and had spikes superimposed, leading to contractions. Desensitization of P2-purinoceptors by alpha,beta-MeATP (3-5 x 10(-6) M) abolished the EJPs and spikes, and reduced the contraction. Atropine (10(-6) M) alone did not alter the EJPs but reduced the contraction. Combined application of both agents abolished the contraction. 6. It is concluded that in the guinea-pig, rabbit and pig bladder ATP is an excitatory transmitter with ACh and EJPs are mediated by ATP.

We developed a prototype apparatus for microexplosion lithotripsy in the urinary bladder according to our original idea. A model calculus in a pig bladder was successfully crushed by the apparatus and was removed by aspiration.