Detrusor Samples Research Articles

Antimuscarinic actions on bladder urothelium and lamina propria contractions are similar to those observed in detrusor smooth muscle preparations.

Antimuscarinic medications are the first-line treatments for overactive bladder, the most common form of bladder dysfunction. Their primary action is thought to block detrusor muscarinic receptors. It is unclear, however, if these therapeutics have actions on other tissues within the lower urinary tract. This study assessed whether clinical antimuscarinics have a functional impact on urothelium with lamina propria (U&LP) tissue. Strips of porcine detrusor and U&LPwere mounted in carbogen-gassed Krebs-bicarbonate solution at 37°C. The tissues were paired with carbachol-response curves performed in the absence or presence of each antimuscarinic. pEC50 values for each curve were analyzed and estimated affinities calculated. Both detrusor and U&LPtissues contracted with muscarinic receptor stimulation, which was inhibited by commonly used antimuscarinics. In detrusor samples (p < 0.001 for all), right parallel shifts from the control were observed in response to oxybutynin (1 µM), solifenacin (1 µM), tolterodine (1 µM), darifenacin (100 nM), trospium (100 nM) and fesoterodine (100 nM). This shift was consistent in U&LPsamples, with no significant differences between the two layers. The data suggests that clinical antimuscarinics are as effective at inhibiting tonic contractions of the U&LPas they are on detrusor, presenting the U&LPas an alternate target of these medications in the treatment of lower urinary tract symptoms.

Contractile function of detrusor smooth muscle from children with posterior urethral valves – The role of fibrosis

Posterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear. We tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. Invitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples. Isometric contractions were measured invitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue. PUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (β-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that invivo would lead to reduced filling compliance. Replacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents.

Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications.

This study aims to characterise the molecular mechanisms that determine variability of atropine resistance of nerve-mediated contractions in human and guinea pig detrusor smooth muscle. Atropine resistance of nerve-mediated contractions and the role of P2X1 receptors, were assessed in isolated preparations from guinea pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve-mediated ATP release was measured directly with amperometric ATP-sensitive electrodes. Ecto-ATPase activity of guinea pig and human detrusor samples was measured in vitro by measuring the concentration-dependent rate of ATP breakdown. The transcription of ecto-ATPase subtypes in human samples was measured by qPCR. Atropine resistance was greatest in guinea pig detrusor, absent in human tissue from normally functioning bladders, and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP-diphosphohydrolase apyrase, directly implicating ATP in their generation. E-NTPDase-1 was the most abundantly transcribed ecto-ATPase of those tested, and transcription was reduced in tissue from human overactive, compared to normal, bladders. E-NTPDase-1 enzymic activity was inversely related to the magnitude of atropine resistance. Nerve-mediated ATP release was continually measured and varied with stimulation frequency over the range of 1-16Hz. Atropine resistance in nerve-mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E-NTPDase-1 activity. ATP is released under different stimulation conditions compared with ACh, implying different routes for their release.

Activation of Prejunctional P2x2/3 Heterotrimers by ATP Enhances the Cholinergic Tone in Obstructed Human Urinary Bladders.

The objective of this study was to investigate the role of ATP in cholinergic neurotransmission in the urinary bladder of control men and of patients obstructed as a result of benign prostatic hyperplasia (BPH). Human detrusor samples were collected from 41 patients who submitted to transvesical prostatectomy resulting from BPH and 26 male organ donors. The release of [3H]acetylcholine ([3H]ACh) was evoked by electrical field stimulation (10 Hz, 200 pulses) in urothelium-denuded detrusor strips. Myographic recordings were performed to test detrusor strip sensitivity to ACh and ATP. Nerve-evoked [3H]ACh release was 1.5-fold higher in detrusor strips from BPH patients compared with controls. This difference was abolished after desensitization of ionotropic P2X1-3 receptors with an ATP analog, α,β-methylene ATP (30 μM, applied for 15 minutes). TNP-ATP (10 nM, a preferential P2X2/3 antagonist) and A317491 (100 nM, a selective P2X3 antagonist) were about equipotent in decreasing nerve-evoked [3H]ACh release in control detrusor strips, but the selective P2X1 receptor antagonist NF023 (3 μM) was devoid of effect. The inhibitory effect of TNP-ATP (10 nM) increased from 27% ± 9% to 43% ± 6% in detrusor strips of BPH patients, but the effect of A317491 (100 nM) [3H]ACh release unaltered (20% ± 2% vs. 24% ± 4%). The amplitude of ACh (0.1-100 μM)-induced myographic recordings decreased, whereas sensitivity to ATP (0.01-3 mM) increased in detrusor strips from BPH patients. Besides the well characterized P2X1 receptor-mediated contractile activity of ATP in pathologic human bladders, we show here for the first time that cholinergic hyperactivity in the detrusor of BPH patients is facilitated by activation of ATP-sensitive P2X2/3 heterotrimers. SIGNIFICANCE STATEMENT: Bladder outlet obstruction often leads to detrusor overactivity and reduced bladder compliance in parallel to atropine-resistant increased purinergic tone. Our data show that P2X1 purinoceptors are overexpressed in the detrusor of patients with benign prostatic hyperplasia. Besides the P2X1 receptor-mediated detrusor contractions, ATP favors nerve-evoked acetylcholine release via the activation of prejunctional P2X2/3 excitatory receptors in these patients Thus, our hypothesis is that manipulation of the purinergic tone may be therapeutically useful to counteract cholinergic overstimulation in obstructed patients.

Chronic high-fat diet decreased detrusor mitochondrial respiration and increased nerve-mediated contractions.

To assess the impact of chronic high-fat diet (HFD) on behavioral voiding patterns, detrusor contractility, and smooth muscle mitochondrial function in male mice. Male C57BL/6J mice (6 weeks) were fed a control or HFD for 20 weeks. Bladder function was assessed by void spot assays. Bladders were collected and detrusor contractility to carbachol (10-9 -10-5 M), and electrical field stimulation (EFS, 0.5-32 Hz) in the presence and absence of atropine was measured. Homogenized detrusor samples were placed in oxygraphs to assess the rate of oxygen consumption of the mitochondria within the detrusor in the presence of different substrates. Mitochondrial hydrogen peroxide (H2 O2 ) emission was measured fluorometrically. Detrusor citrate synthase activity was measured via enzyme activity kit and Western blots assessed the electron transport chain (ETC) protein content. HFD significantly increased body weight, adiposity, and blood glucose levels. HFD mice demonstrated increased voiding frequency and increased EFS-induced detrusor contractility. There were no changes in detrusor relaxation or cholinergic-medicated contraction. Mitochondrial respiration was decreased with HFD and H2 O 2 emission was increased. The relative amount of mitochondria in the detrusor was similar between groups. However, ETC complexes V and III were increased following HFD. Chronic HFD increased adiposity, lead to more frequent voiding, and enhanced EFS-mediated detrusor contractions. Mitochondrial respiration was decreased and H2 O 2 emission increased following HFD. Further research is required to determine if alterations in mitochondrial function could play a role in the development of HFD-induced bladder dysfunction.

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA)

PurposeMuscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis – BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. Material and methodsParaffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (Gαq/11, Gαs, Gαi) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor®488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. ResultsConfocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50–60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. ConclusionPLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic “receptor – G protein – promiscuity” and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.

Involvement of calcium regulating ion channels in contractility of human isolated urinary bladder.

This study specified the role of several key calcium-operating ion channels in contraction/relaxation of human detrusor muscle as possible target for overactive bladder (OAB) treatment. Detrusor samples, obtained from 18 males (average age 61.5 ± 5.9 years), were investigated by organ tissue bath method with following agents: diltiazem for L-type voltage-gated calcium channels; 3-fluropyridine-4-carboxylic acid (FPCA) for Orai-STIM channels; SKF 96365-hydrochloride for transient receptor potential (TRP) channels, T-type channels and Orai-STIM channels; 2- aminoethoxydiphenyl borate (2-APB) for inositol-triphosphate receptors (IP3Rs) and Orai-STIM channels. Oxybutynin and mirabegron were tested under the same conditions as controls. Mirabegron, 2-APB and FPCA exhibited the best suppressive effect on carbachol-induced detrusor contractility. As expressed by area under the contractile curve (AUCC), 2-APB, FPCA and mirabegron have similar AUCC: 1.79, 1.73, 1.73. The highest AUCC was 3.64 for diltiazem+SKF, followed by 3.21 for diltiazem, 3.16 for SKF and 2.94 for oxybutynin. The lowest median amplitude and contraction variability is for 2-APB followed by mirabegron and FPCA. There were significant differences between: 2-APB/FPCA vs.: ditiazem, diltiazem+SKF and SKF. Summary of results suggested the principal role of IP3Rs, Orai-STIM coupling and large-conductance calcium-activated potassium channels in detrusor contraction and pointed on Orai-STIM channels as possible targets for OAB pharmacotherapy.

A simple, rapid, and efficient method for isolating detrusor for the culture of bladder smooth muscle cells.

To establish a simple and rapid method to remove serosa and mucosa from detrusor for the culture of bladder smooth muscle cells (SMCs). Fourteen New Zealand rabbits were randomly allocated to two groups. In the first group, pure bladder detrusor was directly obtained from bladder wall using novel method characterized by subserous injection of normal saline. In the second group, full thickness bladder wall sample was cut down, and then, mucosa and serosa were trimmed off detrusor ex vivo. Twelve detrusor samples from two groups were manually minced and enzymatically digested, respectively, to form dissociated cells whose livability was detected by trypan blue exclusion. Proliferative ability of primary culture cells was detected by CCK-8 kit, and purity of second-passage SMCs was detected by flow cytometric analyses. Another two detrusor samples from two groups were used for histological examination. Subserous injection of normal saline combined with blunt dissection can remove mucosa and serosa from detrusor layer easily and quickly. Statistical analysis revealed the first group possessed higher cell livability, shorter primary culture cell doubling time, and higher purity of SMCs than the second group (P < 0.05). Histological examination confirmed no serosa and mucosa existed on the surface of detrusor obtained by novel method, while serosa or mucosa residual can be found on the surface of detrusor obtained by traditional method. Pure detrusor can be acquired from bladder wall conveniently using novel method. This novel method brought about significantly higher purity and cell livability as compared to traditional method.

Age-related decrease of adenosine-mediated relaxation in rat detrusor is a result of A2B receptor downregulation.

To analyze the effect of adenosine on detrusor smooth muscle contraction and to assess age-related changes of adenosine function. Sustained contractions were induced in young (10-30 days) and old (>60 days) rat detrusor muscle strips by application of 30 mmol/L K(+) and adenosine (0.1-400 µmol/L), which was either applied before raising the K(+) concentration or added to the precontracted muscle strip. Quantitative polymerase chain reaction analyses were used to study adenosine receptor expression in rat and human detrusor specimens. Pretreatment with adenosine dose-dependently reduced subsequent K(+) -induced contraction in detrusor muscle strips from young rats (half-maximal effect = 40 µmol/L). The residual depolarization-induced contraction strength in young tissue was significantly smaller than in tissue from old animals, showing a greater potency of adenosine in young detrusor samples. Likewise, the relaxing effect of adenosine on precontracted detrusor muscle was also significantly more pronounced in young compared with older detrusor. Quantitative polymerase chain reaction showed an age-related downregulation of the adenosine A2B receptor in rat detrusor tissues, which could be confirmed in human detrusor samples. Furthermore, relaxation of both K(+) -induced as well as carbachol-induced contraction by the specific A2B receptor agonist BAY 60-6583 was significantly more pronounced in young than in old rats. Adenosine powerfully counteracts contraction of detrusor smooth muscle, which is lost in the aging bladder. This is paralleled by an age-dependent transcriptional downregulation of the low-affinity A2B receptor. Hence, this might be pathophysiologically relevant in conditions of raised adenosine concentrations, such as hyperactive bladder contractility.

Effect of Age on the Role of Rho-Kinase in Short-term Partial Bladder Outlet Obstruction

We examined the expression of Rho-kinase (ROK) isoforms in young and old rabbits' detrusor smooth muscles (SM) during the progression of short-term partial bladder outlet obstruction and correlated them with the time course of obstruction. We obtained detrusor samples from bladders after 1, 3, 7, and 14 days of obstruction and also sham-operated control rabbits. We used reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting to determine the relative levels of ROK isoform expression at the mRNA and protein levels. Bladder weight for young rabbits increased between 1 and 7 days' obstruction and came back toward control levels at 14 days' obstruction. In old rabbits, bladder weight increased after obstruction, reaching a maximum at 3 days and remaining at this level throughout the 14 days. In young rabbits, the expression of ROKalpha increased in 1- to 7-day obstructed groups and decreased in the 14-day group, whereas it increased progressively in the old rabbits at both the mRNA and protein levels. There was a significant decrease in the expression of ROKbeta in young obstructed rabbits, which gradually decreased during the course of 1- to 7-day obstruction period and increased after 14 days of obstruction. In old groups, there was a decrease in expression after 1 day of obstruction and values remained at a decreased level throughout the course of the study. Young rabbit bladders are better able to adapt to bladder outlet obstruction and ROK isoforms respond in a similar way.

Long term partial bladder outlet obstruction induced contractile dysfunction in male rabbits: A role for Rho‐kinase

In this study we examined the expression of Rho-kinase (ROK) isoforms in rabbit detrusor smooth muscle during the progression of partial bladder outlet obstruction and correlated them with the time course of obstruction. Detrusor samples were obtained from bladders after 1, 2, 4, and 8 weeks of obstruction and also sham operated control rabbits. Contractile responses to field stimulation (FS) and also the smooth muscle (SM) to collagen ratio were determined in isolated bladder strips. Reverse transcriptase-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were used to determine the relative levels of ROK isoform expression at the mRNA and protein levels. Bladder weight increased gradually and contractile responses were reduced significantly over the course of obstruction. The smooth muscle/collagen ratio increased significantly during the course of obstruction. The expression of ROKalpha increased significantly to approximately the same extent in 1-4-week obstructed groups and increased further in the 8-week obstructed group, both at the mRNA and protein levels. In contrast, there was a significant decrease in the expression of ROKbeta in the obstructed groups, which gradually decrease during the course of 1-4-week obstruction period and are slightly upregulated at the decompensated stage at 8-week obstruction. The change in the isoforms of ROK may be part of the molecular mechanism for bladder compensation following partial bladder outlet obstruction.

Relaxation of Phasic Contractile Activity of Human Detrusor Strips by Cyclic Nucleotide Phosphodiesterase Type 4 Inhibition

ObjectivesDetrusor smooth muscle relaxation is mainly mediated by the cyclic adenosine monophosphate (cAMP) pathway. Elevation of cAMP levels by phosphodiesterase type 4 (PDE4) inhibition relaxes smooth muscles of various origins. We aimed to determine the effect of a PDE4 inhibitor, rolipram, on human detrusor contractions. MethodsHuman bladder strips (from 20 different donors) with no known overactive bladder (OAB) were studied in organ baths. Detrusor samples with or without urothelium were incubated with carbachol 10−6mol/l (in presence or absence of forskolin, 3.10−7mol/l) or with KCl 10mmol/l to enhance phasic contractile activity. Concentration response curves for rolipram or vehicle were then performed. ResultsRolipram (10−9 to 3.10−5mol/l) induced a moderate relaxing effect on carbachol-induced contractions. This effect was enhanced when cAMP levels were increased by forskolin (the maximal effect was 53.0±5.1 vs. 83.1±5.7%, p<0.01) or in strips with urothelium. In contrast, rolipram (10−9 to 10−4mol/l) drastically inhibited phasic contractile activity: The developed tension, the area under the curve, and the amplitude of phasic activity were reduced to 64.8±3.6, 91.2±5.3, and 82.3±7.3%, respectively, versus 23.6±9.5, 34.7±18.8, and 18.0±16.2% for vehicle, respectively (p<0.05). Frequency of phasic activity was 0.96±0.45 contractions per minute versus 2.6±0.18 for vehicle (p<0.001). In strips with urothelium, the inhibitory effect of rolipram on phasic contractile activity was similar. ConclusionsPDE4 isoenzymes are strongly involved in the regulation of phasic myogenic activity of human bladder strips. Because an increase of this phasic activity may play a role in the pathophysiology of detrusor overactivity, PDE4 inhibitors might represent an attractive strategy for the treatment of OAB.

Expression of Smoothelin in the Normal and the Overactive Human Bladder

We established the expression pattern of smoothelin, a marker protein for contractile smooth muscle cells, in the human detrusor and investigated its possible impact on bladder overactivity. Detrusor samples of 13 overactive bladders (sensory urge and detrusor instability) were obtained before botulinum toxin injection and compared to those of 8 normally contractile, nonobstructed bladders obtained during radical cystectomy. Smoothelin mRNA expression patterns were investigated by Northern blot and variant specific reverse transcriptase-polymerase chain reaction as well as by quantitative reverse transcriptase-polymerase chain reaction on laser capture, microdissected smooth muscle. At the protein level smoothelin was investigated by standard and quantitative immunohistochemistry. The bladder muscularis expressed vascular and visceral smoothelin isoforms, and 2 of the known splice variants. In the smooth muscle of patients with detrusor instability and sensory urge a significant 2.4 and 2.2-fold increase, respectively, in smoothelin variant 1 mRNA was observed in comparison to that of normal controls. Analyses at the smoothelin protein level confirmed significant up-regulation in these bladder dysfunctions by a factor of 2.3 and 1.8, respectively. No significant difference in smoothelin expression was observed between detrusor instability and sensory urge. Increased expression of smoothelin in patients with detrusor instability and sensory urge implies that the etiology of these dysfunctions includes changes in myogenic parameters. In addition, our data support the new classification of the International Continence Society for overactive bladder proposing that sensory urge and detrusor instability represent a single clinical entity.

Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency.

Purinergic P2X receptors associated with the parasympathetic nerves supplying human bladder smooth muscle (detrusor) are implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalised with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder and in adults with the urodynamics findings of sensory urgency (SU) to determine how receptor distribution varied in patients with a small bladder capacity. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody (SV2) against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for haematuria (n=22, age 33-88 years) and adults with sensory urgency at cystoscopy/cystodistension (n=11, age 37-70 years). Normal adult specimens contained detrusor muscle innervated by parasympathetic nerves possessing large varicosities (1.2 microm) distributed along their length. These mostly all showed colocalised patches of presynaptic P2X(1,2,3,5) subtypes while presynaptic subtypes P2X(4,6,7) were present in only 6-18% of varicosities. Detrusor nerve varicosities from SU patients revealed general loss of all presynaptic P2X subtypes with the proportion containing receptors reducing to only 0.5-5% depending on P2X subtype. The same loss was recorded from the sensory nerves in the surrounding lamina propria. This specific loss of P2X receptors may impair control of detrusor distension and contribute to the pathophysiology of sensory urgency.

Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders.

Three of this month's Scientific Discovery papers highlight the importance of collaboration in delivering high quality scientific research. As scientific technology increases in power and cost, and specific areas of interest become more specialized, it is becoming more difficult to cover all aspects of a completeresearch story. Collaborating with other experts in the field or other fields, including industry, allows strong scientific proof to be generated for the hypothesis and aims. Building strong collaborative,inter-disciplinary, multi-institutional, international groups with academic and industrial partners is the way forward for all discovery. We look forward to publishing more of these collaborative papersin future issues of the BJU International. To test the hypothesis that intercellular electrical coupling is altered in human detrusor smooth muscle from patients with unstable bladders. Human detrusor biopsy samples were obtained from patients with stable and unstable bladders. Intracellular electrical impedance was measured with alternating current (20 Hz-300 kHz) across the ends of detrusor strips in an oil-gap, after correcting for extracellular space resistance. Gap junctions were identified by localization of connexins (Cx), specifically Cx45, Cx43 and Cx40 transcripts, using immunoconfocal microscopy. Total intracellular resistivity was greater in strips from unstable than from stable bladders (median 1246 vs 817 Omega.cm). The increase was attributed to an increase in junctional resistance; cytoplasmic resistance was unchanged. Cx43 was localized to a submucosal layer and to connective tissue; Cx40 label was confined to endothelial cells of blood vessels. Cx45 labelling was localized to detrusor bundles and appeared to be less marked in samples from unstable bladders. Semi-quantitative analysis of Northern blots showed that Cx45 expression in unstable was less than that in stable bladders. These data suggest that intercellular coupling is reduced in detrusor from unstable bladders. Cx45 was localized to the detrusor layer, with Cx 43 more evident in the suburothelial mucosa. Cx45 labelling was less intense in detrusor samples from unstable bladders. These results are consistent with reduced gap junction coupling in detrusor from unstable bladders.

Loss of Purinergic P2X3and P2X5Receptor Innervation in Human Detrusor from Adults with Urge Incontinence

Activation of purinergic P2X receptors associated with the parasympathetic nerves that supply the human bladder smooth muscle (detrusor) is implicated in control of detrusor contractility. The relative abundance of all seven subtypes colocalized with synaptic vesicles on parasympathetic nerves was examined in specimens from normal adult bladder, infants, and in adults with overactive detrusor contractility and a diagnosis of idiopathic detrusor instability (IDI) to determine whether receptor distribution varied with age or in patients with incontinence. Alteration in control of detrusor innervation was examined with P2X subtype-specific antibodies and an antibody against synaptic vesicles, using immunofluorescence and confocal microscopy. Detrusor samples were taken from: controls, at cystectomy for cancer or cystoscopic biopsy for hematuria (n = 22; age 33-88), child bladder, at surgical correction of vesico-ureteric reflux (n = 21; age 4 months to 2 years), and adults with detrusor instability at cystoscopy-cystodistension (n = 18; age 30-81). Adult specimens contained muscle with large varicosities (1.2 microm) along parasympathetic nerves with colocalized patches of all P2X(1-7) subtypes. Infant bladder revealed little evidence of P2X at age <9 months but approached adult levels at 2 years. Detrusor from IDI patients revealed selective absence of P2X(3) and P2X(5) beneath all the varicosities. This specific lack of P2X(3) and P2X(5) may impair control of detrusor contractility and contribute to the pathophysiology of urge incontinence.

A QUANTITATIVE STUDY OF ATROPINE-RESISTANT CONTRACTILE RESPONSES IN HUMAN DETRUSOR SMOOTH MUSCLE, FROM STABLE, UNSTABLE AND OBSTRUCTED BLADDERS

The objective of the study was to quantify in vitro the magnitude of atropine-resistant contractions using human detrusor samples and to determine the cellular processes underlying these contractions. Isometric contractile responses were measured in isolated strips of human detrusor muscle obtained from patients with i) stable, ii) unstable or iii) obstructed bladders. Preparations were electrically stimulated or exposed to carbachol and ATP in the superfusate. Force-frequency curves were shifted to the right in samples from unstable and obstructed bladders. These same tissue groups also showed significant atropine-resistant contractions which were abolished by the neurotoxin TTX, or the non-hydrolysable ATP analog, alpha,beta-methylene ATP, suggesting that these contractions were mediated by neurally released ATP. Sub-division of the patient group with unstable bladders demonstrated that those with neuropathic instability did not show atropine-resistance, whereas those with idiopathic instability or secondary instability after obstruction did show atropine-resistant contractions. The potency of carbachol in generating a contracture was significantly greater than ATP (mean EC50 0.65 microM and 151 microM respectively) however, for each agonist there was no difference in potency between the three patient groups. Direct muscle excitability was similar in all three patient groups. It is concluded that purinergic, atropine-resistant contractions are present in some types of dysfunctional bladder, and these are not caused by a differential sensitivity of the muscle to ATP and cholinergic agonists.

The Unstable Bladder

The unstable bladder is a common clinical problem of uncertain etiology. Current in vitro studies of unstable human detrusor samples show differences in behavior and response to pharmacologic agents from normal detrusor. The fundamental urodynamic abnormality is the occurrence of involuntary detrusor contractions, which cause the cardinal symptom of urgency. The incidence of other symptoms is determined by the functional bladder capacity and the patient's ability to resist the unstable contractions. Having excluded outflow obstruction as a causative or associated factor, empirical treatment may reasonably be started on the basis of the findings of a 48-hour voided volume chart, reserving full urodynamic investigation for patients who fail to respond to treatment as expected. For those with minimal urodynamic dysfunction, bladder drill is the treatment of choice; when this fails or is inappropriate, drug treatment with oxybutynin is indicated, supplemented by other drugs when appropriate. When these standard conservative measures fail, then transvesical injection of the pelvic plexuses with phenol gives worthwhile results and is a trivial procedure. If this fails, and in men in whom the phenol procedure is contraindicated, "clam" ileocystoplasty is usually if not always curative.