Characteristics of Spontaneous Activity in the Bladder Trigone

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1 EUROPEAN UROLOGY 56 (2009) available at journal homepage: Voiding Dysfunction Characteristics of Spontaneous Activity in the Bladder Trigone Alexander Roosen a,d, Changhao Wu b, *, Guiping Sui a, Rasheda A. Chowdhury c, Pravina M. Patel c, Christopher H. Fry a a Department of Surgery, University College London, London, United Kingdom b Department of Medicine, University College London, London, United Kingdom c Department of Cardiac Electrophysiology, National Heart and Lung Institute, Imperial College, London, United Kingdom d Department of Urology, Ludwig-Maximilians-University, Muenchen, Germany Article info Article history: Accepted June 12, 2008 Published online ahead of print on June 20, 2008 Keywords: Trigone Spontaneous activity Connexin43 L-type Ca 2+ -channels Cl -channels Guinea-pig Abstract Background: During bladder filling, the trigone contracts help keep the ureteral orifices open and the bladder neck shut. The trigone generates spontaneous activity as well as responding to neuromuscular transmitters, but the relationship between these phenomena are unclear. Objectives: To characterise the cellular mechanisms that regulate and modify spontaneous activity in trigone smooth muscle. Design, setting, and participants: Muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments and immunofluorescent studies. Measurements: In isolated trigonal cells, intracellular Ca 2+ was measured by epifluorescence microscopy using the fluorescent Ca 2+ indicator Fura-2. Results and limitations: Spontaneous intracellular Ca 2+ transients and contractions were observed in trigonal single cells and strips and were significantly higher compared to the bladder dome. Ca-free superfusate and verapamil terminated spontaneity. T-type Ca 2+ channel block with NiCl 2 depressed slightly Ca 2+ transients but not spontaneous contractions. Neither the BK Ca channel blocker iberiotoxin nor the SK Ca channel blocker apamin had any effect on single cell activity. By contrast, the Cl channel blocker niflumic acid attenuated significantly both Ca 2+ transients and muscle contractions. Agonist stimulation (carbachol, phenylephrine) up-regulated activity. Gap junction labelling (Cx43) was approximately 5 times denser in the trigone than in detrusor smooth muscle. The gap junction blocker 18-ß-glycyrrhetinic acid modulated spontaneous contractions in the trigone but not in the bladder dome. Conclusions: Trigone myocytes employ membrane L-type-Ca 2+ channels and Cl channels to generate spontaneous activity. Intercellular electrical coupling ensures its propagation and, thus, sustains contraction of the whole trigone. # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding author. Postgraduate Medical School, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7WG, UK. Tel address: c.wu@surrey.ac.uk (C. Wu) /$ see back matter # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eururo

2 EUROPEAN UROLOGY 56 (2009) Introduction The trigone has recently attracted urologists attention again, initiated by a discussion of whether it should be spared in intradetrusor botulinum toxin injections to treat bladder overactivity [1,2]. It is strategically located between the ureteric orifices and bladder outlet; however, little is known about its function during the micturition cycle. The superficial trigone develops, with the ureter, from an outgrowth of the mesonephric duct and provides, as a transverse-orientated interureteric muscle, competent vesico-ureteric anchoring [3,4]; it represents an area of dual parasympathetic-muscarinic and sympathetic-adrenergic innervation. There is evidence that micturition is initiated by relaxation of the trigone, which then funnels urine into the proximal urethra; relaxation may be NO/ cgmp-mediated through nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase positive nerves [5,6]. However, information about trigone behaviour during filling is sparse, and spontaneous activity might play a crucial role. Various smooth muscles in the lower urinary tract show spontaneous contractile activity during the filling phase. In the bladder dome, this behaviour may help the organ maintain a state of minimal surface area; in the urethra, it contributes significantly to closure pressure [7]. Although spontaneous activity has been intensively investigated in both dome and urethra, studies in the trigone are lacking. Two papers, which primarily investigated agonist effects on detrusor muscle, noted pronounced spontaneous activity of the trigone. Contractile activity was present in 71% and 89% of trigone strips from pigs and humans, respectively, compared to 20% from the dome [8]. Microelectrode recordings showed rhythmic variation of membrane potential in the dome; but in the trigone, there were superimposed additional bursts of spikes [9]. This study also described up-regulation of trigone activity by agonist stimulation. We set out to corroborate previous reports of spontaneous activity in the trigone, to elucidate its origin and physiological characteristics, and to explore possible routes of modulation at an intra- and intercellular level. Data were compared to those from similar detrusor preparations of the bladder dome. 2. Methods 2.1. Tissue preparation and tension experiments Tissue was obtained from the bladders of 70 male guinea-pigs ( g, Dunkin-Hartley), killed by Schedule-1 cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act, The bladder dome was resected cranially to the orifices, the base longitudinally opened on the ventral site, and the trigonal area exposed. The superficial layer was distinguished from underlying detrusor by its paler, whitish appearance. After removing the mucosa by blunt dissection, a thin strip (average: 6.4 mg) between the ureteral orifices was cut; only one strip per bladder was prepared. Strips were also dissected from the dome. Preparations (28 trigone, 21 dome) were mounted in a horizontal superfusion trough between a fixed hook and an isometric force transducer and superfused with a 24 mm-nahco 3 /5%CO 2 -buffered Tyrode s solution (5 10 ml min 1, 378C, ph 7.4). All chemicals throughout were from Sigma unless specified in the text. Transducer output was recorded through a bridge amplifier and displayed on a moving-paper chart recorder Cell isolation, measurement of intracellular calcium ([Ca 2+ ] i ) Cells were prepared from muscle strips (37 animals) as might be used for tension measurements by dissociation with a collagenase-based enzyme mixture [10]. Cells were loaded with the fluorescent Ca 2+ indicator Fura- 2 (Molecular Probes) at 37 8C for 9 min and stored at 4 8C for later use. An aliquot of cell suspension was placed in a glass-bottomed chamber maintained at 37 8C, mounted on the stage of an inverted microscope. After cells had settled to the chamber floor, it was superfused with Tyrode s at 1.5 ml min 1. Cells were illuminated alternately at 340:380 nm (32 Hz) and fluorescent light collected between 410 and 510 nm with a photomultiplier tube. The ratio of fluorescence on excitation at 340 or 380 nm is a function of [Ca 2+ ] i : Calibration of the signal has been detailed elsewhere [10] Connexin immunofluorescence Five de-urothelialised strips were placed immediately in optimal cutting temperature (OCT) embedding medium and snap-frozen in liquid N 2. Triplicate sections (10 mm) for each specimen were cut on a cryostat and mounted on 3-aminopropyltriethoxysilane (APES)-coated slides. Sections were post-fixed in methanol ( 20 8C, 5 min), blocked in 1% bovine serum albumin (BSA) for 45 min and incubated with primary antibodies against connexin43 and connexin45 (2 h, room temperature: Cx43, mouse mab, Chemicon 1:1000; Cx45; gift from Professor N Severs, Imperial College London). Binding was visualised using a Cy3- conjugated secondary antibody (goat, anti-mouse, Chemicon, 1:1000), and nuclei counterstained with 4 0,6-diamidino-2-phenylindole (DAPI; Invitrogen, 1:50,000) in the same step (45 min, room temperature [RT]). Three representative images per section were taken using a confocal microscope (Zeiss LSM 510 Meta, x40) at fixed pinhole and detector gain settings. The number of punctate connexin fluorescent particles was counted in each image using the ImageJ freeware program (rsb.info.- nih.gov/ij/; constant threshold, particles 2-infinity) Experimental protocols and data analysis Experimental variables obtained during interventions were compared to the mean of pre- and postintervention values (controls) and expressed either as absolute values or percentage of control. Control experiments, using only the vehicle for test agents, were always carried out and exerted no significant effects. Values are mean SD, n = number of cells or muscle strips. Differences between means of data sets were examined by paired or unpaired student t tests and those between two incidences by Fisher s exact test. The null hypothesis was rejected at p < Results 3.1. Characteristics of spontaneous activity Spontaneous activity was recorded in trigonal single cells and strips as Ca 2+ transients and contractions, respectively. Myocytes were about half the size of those from the bladder dome (Fig. 1A). The resting [Ca 2+ ] i was nm (n = 107 cells), and cells always showed either discrete Ca 2+ transients (Fig. 1B) or, less often, more sustained and fused

3 348 EUROPEAN UROLOGY 56 (2009) increases of [Ca 2+ ] i (Fig. 1C). Their mean amplitude was nm. Cells regularly responded with visible contractions to Ca 2+ transients (Fig. 1A, B and C: c), demonstrating their functional relevance. Furthermore, the majority of strips generated regular contractions after 45-min equilibration (Fig. 1D) mean amplitude mn mm 2 (75.3% and 207.1% of force generated by 10 mm phenylephrine [PE] and 1 mm carbachol, respectively). Preparations not generating phasic contractions always displayed uncoordinated baseline changes (Fig. 1E). The incidence of activity in the trigone was significantly higher than in the dome. Only eight of 21 strips (38%) from the dome showed spontaneous contractions after 45 min, whereas most trigone strips (19 of 28, 68%) developed them. The mean frequency of Ca 2+ transients and contractions was greater in the trigone compared to detrusor: Ca 2+ transients vs min 1 (n = 40,6; amplitude vs nm); contractions vs min 1 (n = 14,6; no significant differences in amplitude) Cellular mechanisms for spontaneous activity Fig. 1 (A) Image of a trigonal myocyte before (upper left) and after (lower left) spontaneous contraction compared to a myocyte from the bladder dome (right) (all images on the same scale); (B and C) representative [Ca 2+ ] i recordings of unstimulated trigonal single cells; (D and E) tension recordings from unstimulated strips. c, contraction parts (A), (B), and (C). Intracellular stores were investigated as a possible source of spontaneous activity. Thapsigargin (1 mm, 20 min, n = 10) and carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP; 4 mm, 20 min, n = 4) were used to block Ca 2+ release from the sarcoplasmic reticulum or mitochondria, respectively: however, no alteration to activity was seen (Fig. 2A). By contrast, the L-type-Ca 2+ channel blocker, verapamil (20 mm, Knoll) or Ca-free solution (zero-added Ca plus 0.5 mm ethylene glycol tetraacetic acid [EGTA], n = 20) abolished activity completely (Fig. 2A, B). Corresponding spontaneous muscle contractions and baseline tension were reduced by verapamil (20 mm, n =6; Fig. 2C) and Ca-free solution (n = 4, Fig. 2D). T-type-Ca 2+ channels may facilitate the opening of L- type channels, as the former are activated at more negative Fig. 2 (A) Representative trace showing the effect of thapsigargin (blocking Ca 2+ release from the SR), FCCP (blocking mitochondrial Ca 2+ release), verapamil (blocking L-type Ca 2+ channels) on spontaneous [Ca 2+ ] i transients; (B) effect of Ca 2+ -free solution on spontaneous [Ca 2+ ] i transients; (C) effect of verapamil on spontaneous contractions; (D) effect of Ca 2+ -free solution on spontaneous contractions; (E) summary of the effects of NiCl 2 and MgCl 2 on the amplitude and frequency of spontaneous [Ca 2+ ] i transients data as % of control, mean W SD, * p < 0.05.

4 EUROPEAN UROLOGY 56 (2009) potentials. Superfusion with 100 mm NiCl 2, to block selectively T-type channels attenuated significantly the frequency and amplitude of Ca 2+ transients: frequency to min 1 ( % control); amplitude to nm ( % control, n = 5) Fig. 2E. However, there was no corresponding effect on muscle strips (n = 4). Magnesium stabilises various smooth muscle tissues by attenuating L-type Ca 2+ current [11]. MgCl 2 (2 mm, 5 min, n = 5) reduced Ca 2+ transient frequency ( to min 1 [69 21% control]) and amplitude ( to nm, [ % control]) Fig. 2E without a significant effect on muscle strips. In detrusor myocytes, large conductance Ca 2+ -activated K + -channels (BK Ca ) are mainly responsible for repolarisation of spontaneous electrical activity, and small conductance K + -channels (SK Ca ) have a modulatory role. Neither the BK Ca -channel blocker iberiotoxin (50 nm) nor the SK Ca - channel blocker apamin (100 nm) had any effect on isolated trigonal cells when applied for up to 20 min (n = 8 and 9, respectively; data not shown). Cl channels can generate spontaneous Ca 2+ transients in distinct cell populations of the lower urinary tract (eg, urethral and bladder interstitial cells) [12,13]. With isolated trigonal cells, the Cl channel blocker niflumic acid (100 mm) attenuated the frequency ( to min 1 [ % control, n = 7]) and amplitude ( to nm [ % control, n =7],Fig. 3A and E) of Ca 2+ transients. Muscle contractions were similarly reduced in frequency and amplitude ( to min 1 [ % control] and to mn mm 2 [ % control], n = 5, Fig. 3C and F). Reducing extracellular Cl shifts the reversal potential of acl current to more positive potentials, depolarises cells, and should increase spontaneous activity. Extracellular Cl was reduced from to 10.7 mm by equimolar replacement of NaCl with Na isethionate, whilst maintaining constant Ca 2+ activity by raising extracellular CaCl 2 from 1.8 to 2.34 mm [14].Ca 2+ transient frequency and amplitude in isolated cells were increased ( to min 1 [ % control] and to nm [ % control, n = 5], Fig. 3B and F). In strips, contraction amplitude was similarly augmented ( to mn mm 2 [ % control, n = 5], Fig. 3D and F), although frequency was not significantly altered ( min 1 vs min 1 ) Modulation of trigonal spontaneity Adrenergic and muscarinic receptor stimulation increases spontaneous activity in detrusor myocytes [9,15]. Trigone myocytes were exposed to 10 mm PE and 1 mm carbachol for 1 min (concentrations exerting intermediate inotropic effects on muscle strips; see also Roosen et al [16]). Both interventions significantly increased the frequency and amplitude of Ca 2+ transients (PE: frequency to min 1 [ % control], amplitude to nm [ %, n = 9]; carbachol: frequency to min 1 [ % control], amplitude to nm [ % control, n = 9]), as Fig. 3 (A) Representative trace showing the effect of the Cl S channel blocker niflumic acid on spontaneous [Ca 2+ ] i transients; (B) effect of low Cl S solution on spontaneous [Ca 2+ ] i transients note the interruption of the trace during the low Cl S intervention; (C) effect of niflumic acid on spontaneous contractions; (D) effect of low Cl S solution on spontaneous contractions; (E and F) summary of the effects of niflumic acid and low Cl S solution on the amplitude and frequency of spontaneous [Ca 2+ ] i transients (E) and spontaneous contractions (F) data as % of control, mean W SD, * p < 0.05.

5 350 EUROPEAN UROLOGY 56 (2009) Fig. 4 (A) Representative trace showing the effect of 10 mm phenylephrine (PE, 1 min) and 1 mm carbachol (carb, 1 min) on spontaneous [Ca 2+ ] i transients; (B) summary of the effects of 10 mm PE and 1 mm Carb on the amplitude and frequency of spontaneous [Ca 2+ ] i transients (measured 3 min after intervention); data as % of control, mean W SD; * p < shown in Fig. 4A and B. In muscle strips, spontaneous activity normally ceased with the development of agonist-induced contractions. Gap junctions electrically couple adjacent cells and are formed from different isoforms of the connexin (Cx) protein family. In the trigone and detrusor, we found labelling for Cx43 (Fig. 5A and B). However, labelling was more intense in the trigone ( vs punctate regions per mm section, n = 4, Fig. 5E). Moreover, in the trigone, labelling was more evenly distributed, whereas in the detrusor it was strictly confined to a chain-like distribution associated with cells that had smaller, long nuclei between muscle bundles (arrows, Fig. 5A). No labelling for Cx45 was observed in the trigone, as has been recorded in the detrusor between adjacent smooth muscle cells. To test the significance of gap junction proteins for spontaneous contractions in trigone and detrusor strips, we perfused them with the gap junction blocker 18-ß-glycyrrhetinic acid (10 mm, 18-ß-GA) for 5 min. Detrusor strips were not significantly affected (frequency: vs min 1 ; amplitude: vs mn mm 2, n = 5, Fig. 5C and F), However, trigonal contractions became significantly more frequent ( vs min 1 [ % control]) and smaller ( vs mn mm 2 [ %, n =5],Fig. 5D and F). In addition, even smaller contractions appeared between larger ones, generating a less-organised contractile pattern. 18-ß-GA had no effect on Ca 2+ transients in trigonal single cells (n =4). 4. Discussion This is the first study to investigate the nature and modulation of spontaneous activity in the trigone. The Ca 2+ transients were comparable to those generated by receptor agonists of the major contractile neurotransmitters PE and carbachol, and thus represent significant events that could support cellular contraction. Cells always responded to both PE and carbachol which differentiated them from detrusor myocytes (respond to carbachol alone) or urethral myocytes (respond to PE alone). The general harmony of observations with isolated cells and muscle strips suggests that Ca 2+ transients in trigone myocytes are the precursor of spontaneous contractions. The source of Ca 2+ for intracellular transients is probably through L-type Ca 2+ channels, as reducing extracellular Ca, the addition of verapamil, or raising extracellular Mg all attenuated activity. However, blocking Ca 2+ release from internal sources had no effect. T-type channels might also contribute, as they are activated at more negative potentials [17] and so facilitate L-type channel opening. Of interest, Cl channels also modulated spontaneous activity, as evidenced by their attenuation with niflumic acid or enhancement by reducing extracellular Cl. However, blockade of BK Ca or SK Ca channels had no effect, which implies that they do not have predominant effects on membrane activity. Furthermore, as trigone myocytes respond to both adrenergic and muscarinic receptor agonists by increasing force [16] and intracellular Ca 2+, both agonists also increased significantly spontaneous activity, although the cellular pathways remain to be elucidated. Practically, monitoring changes to Ca 2+ transients in isolated cells was a more sensitive index than changes to spontaneous contractions. Most likely, this is because the latter are a summation of asynchronous behaviour in many individual cells so that relatively uncoordinated changes in myocytes would manifest themselves less distinctly in multicellular preparations. The cellular origin of spontaneous activity in the trigone shows differences from other lower urinary tract cells that demonstrate similar activity. In the detrusor, spontaneous contractile activity is associated with action potentials whose upstroke is carried by L-type Ca 2+ current and repolarised largely by BK Ca channel activity [7,15,18]. Transmembrane Ca 2+ fluxes may be supplemented by Ca 2+ release from internal stores, which are replenished through a feedback mechanism employing BK Ca and L-type Ca 2+ channels [10]. In other regions of the urinary tract, such as the renal pelvis and urethra, electrically active atypical

6 EUROPEAN UROLOGY 56 (2009) Fig. 5 (A and B) Representative sections (10 mm) through a muscle preparation from the bladder dome (A) and the trigone (B), labelling for connexin 43 (red) and nuclei (blue) note the sparse, chain-like distribution of Cx43 in the detrusor along a band of spindle-shaped nuclei, presumably interstitial cells (yellow arrows); (C and D) representative tension recordings showing the effect of gap junction-blocker 18-ß-glycyrrhetinic acid (18-ß-GA, 10 mm) on spontaneous contractions of strips from the bladder dome (C) and the trigone (D); (E) summary of gap junction counts per 230 T 230-mm field in the detrusor and trigone; (F) summary of the effect of 18-ß-GA on the amplitude and frequency of spontaneous contractions in strips from the bladder dome and trigone; data as % of control, mean W SD; * p < smooth muscle cells [19] or interstitial cells [12] may drive or modulate spontaneous activity in true muscle cells. Where studied, they operate through a combination of Ca 2+ release from internal stores and opening of depolarising Ca 2+ -activated Cl channels [20], and Cl channel blockers have a marked depressant effect [12,13]. However, K + channels have not been reported to be of particular importance, and Ca 2+ channels are of lower density than in true muscle cells. Both spontaneous [Ca 2+ ] i rises and Ca 2+ -activated Cl channels are characteristic of suburothelial myofibroblasts that also have little L-type Ca 2+ current [21]. Therefore, trigonal myocytes seem to have physiological characteristics intermediate between detrusor myocytes and other cells of the urinary tract, including interstitial cells or atypical renal pelvis cells. The difference between the trigone and the bladder dome was also reflected in gap junction make-up and distribution. The trigone throughout was characterised by

7 352 EUROPEAN UROLOGY 56 (2009) Cx43 labelling, whereas this was confined to labelling between muscle bundles in the detrusor (see also Ikeda et al [22]). Cx45 was not detected in the trigone but comprises intermuscular connections in the detrusor [23]. The dense distribution of trigonal Cx43 is in line with an electronmicroscopic (EM) study in humans suggesting marked electrical coupling in the trigone [24]. The relevance to spontaneous muscle contractions was demonstrated by the effects of the gap junction blocker 18-ß-GA, which reduced the amplitude but increased the frequency of spontaneous contractions. Such a pattern might be expected from reduced intercellular coupling so that spontaneous activity originating in individual cells would be less well coordinated throughout the tissue mass. The lack of effect of 18-ß-GA on detrusor strips is consistent with a lower gap junction density, so that spontaneous activity must already be poorly co-ordinated, and is consistent with 18-ß-GA being ineffective on spontaneous activity of normal rat bladder [22]. It can be hypothesised, therefore, that electrical coupling in the trigone is better than in the detrusor, although still less than in extensively coupled tissues such as myocardium. The relative difference of electrical coupling will have physiological consequences. Poorer coupling in the bladder dome prevents extensive propagation of electrical signals and allows individual muscle bundles to adjust their length in response, say, to a volume change without synchronous activation of the muscle mass that would elevate intravesical pressure ( micromotions, [7]). The trigone is believed to contract during the filling phase, thus contributing to opening the ureteric orifices and closing the bladder outlet. The high spontaneous activity and extensive electrical coupling may help the trigone adopt a state of sustained contracture during urine storage, augmented by neurotransmitter stimulation. Further structural and functional studies should reveal whether trigonal muscle cells display a phenotype more reminiscent of bladder smooth muscle or interstitial cells or even ureter. Homogenous positive labelling for alpha smooth muscle actin (a-sma) has been shown [4,25], but labelling for c-kit and vimentin which have both been proposed as markers for interstitial cells in the lower urinary tract [26,27] would be valuable. Furthermore, the ability of verapamil to abolish spontaneous activity in the ureter [28] was mirrored here, and further comparison between these tissues would be useful. Finally, the present study focused on intrinsic tension generation in the trigone, but an investigation of relaxing mechanisms would also be valuable. NO has been demonstrated as a key factor [6]. Whether it suppresses spontaneous depolarisations of the membrane through the NO/cGMP/PKG pathway as in functionally related interstitial cells of the urethra [29] or lowers Ca 2+ sensitivity of the contractile machinery is unknown. 5. Conclusion Trigonal myocytes show marked spontaneous activity in the form of [Ca 2+ ] i transients resulting from transmembrane Ca 2+ influx through L-type Ca 2+ channels. As with lower urinary tract interstitial cells, Cl channels rather than K + channels also contribute to the generation of spontaneity. Extensive gap junction coupling ensures electrical propagation and sustained spontaneous contraction of the whole trigone, thus contributing to its proposed physiological functions. Author contributions: Alexander Roosen, Changhao Wu and Christopher H. Fry had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy take of the data analysis. Study concept and design: Roosen, Sui, Wu, Fry. Acquisition of data: Roosen. Analysis and interpretation of data: Roosen, Wu, Fry. Drafting of the manuscript: Roosen. Critical revision of the manuscript for important intellectual content: Wu, Fry. Statistical analysis: Roosen, Wu. Obtaining funding: Roosen, Wu, Fry. Administrative, technical, or material support: Chowdhury, Patel. Supervision: Sui, Wu, Fry. Other (specify): none. Financial disclosures: I certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: none. Funding/Support and role of the sponsor: A. Roosen is recipient of a grant of the German Research Foundation. Wellcome Trust and British Heart Foundation provided financial support. Acknowledgement statement: We acknowledge the support of the Deutsche Forschungsgemeinschaft, the Wellcome Trust, and the British Heart Foundation who have no commercial interest in the data. References [1] Karsenty G, Denys P, Amarenco G, et al. Botulinum toxin A (Botox 1 ) intradetrusor injections in adults with neurogenic detrusor overactivity/neurogenic overactive bladder: a systematic literature review. Eur Urol 2008;53: [2] Patel AK, Patterson JM, Chapple CR. Botulinum toxin injections for neurogenic and idiopathic detrusor overactivity. A critical analysis of results. Eur Urol 2006;50: [3] Tanagho EA. The ureterovesical junction. In: Chisholm GD, Williams DI, editors. Scientific Foundations of Urology. London: Heinemann; p [4] Oswald J, Schwentner C, Lunacek A, et al. Reevaluation of the fetal muscle development of the vesical trigone. J Urol 2006;176: [5] Andersson KE, Alm P. Neurogenic nitric oxide and the lower urinary tract. In: Toda N, Moncada S, Furchgott RF, Higgs EA, editors. Nitric Oxide and Peripheral Nervous System. London: Pergamon; p [6] Persson K, Alm P, Johansson K, Larsson B, Andersson KE. Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry and functional effects. Br J Pharmacol 1993;110: [7] Brading AF. Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function. J Physiol 2006;570:13 22.

8 EUROPEAN UROLOGY 56 (2009) [8] Sibley GN. A comparison of spontaneous and nerve-mediated activity in bladder muscle from man, pig and rabbit. J Physiol 1984;354: [9] Callahan SM, Creed KE. Electrical and mechanical activity of the isolated lower urinary tract of the guinea-pig. Br J Pharmacol 1981;74: [10] Wu C, Sui G, Fry CH. The role of the L-type Ca 2+ channel in refilling functional intracellular Ca 2+ stores in guinea-pig detrusor smooth muscle. J Physiol 2002;538: [11] Montgomery BS, Thomas PJ, Fry CH. The actions of extracellular magnesium on isolated human detrusor muscle function. Br J Urol 1992;70: [12] Sergeant GP, Hollywood MA, McCloskey KD, Thornbury KD, McHale NG. Specialised pacemaking cells in the rabbit urethra. J Physiol 2000;526: [13] Sergeant GP, Hollywood MA, McHale NG, Thornbury KD. Ca 2+ signalling in urethral interstitial cells of Cajal. J Physiol 2006; 576: [14] Fry CH, Langley SM. Ion-selective Electrodes in Biomedical Systems. 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Activation of Ca 2+ -activated Cl current by depolarizing steps in rabbit urethral interstitial cells. Am J Physiol Cell Physiol 2003;285:C [21] Wu C, Sui GP, Fry CH. Purinergic regulation of guinea pig suburothelial myofibroblasts. J Physiol 2004;559: [22] Ikeda Y, Fry C, Hayashi F, Stolz D, Griffiths D, Kanai A. Role of gap junctions in spontaneous activity of the rat bladder. Am J Physiol Renal Physiol 2007;293:F [23] Sui GP, Coppen SR, Dupont E, et al. Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders. BJU Int 2003;92: [24] John H, Hauri D, Bangerter U, Elbadawi A. Ultrastructure of the trigone and its functional implications. Urol Int 2001;67: [25] Thomson AS, Dabhoiwala NF, Verbeek FJ, Lamers WH. The functional anatomy of the ureterovesical junction. Br J Urol 1994; 73: [26] Hashitani H, Yanai Y, Suzuki H. Role of interstitial cells and gap junctions in the transmission of spontaneous Ca 2+ signals in detrusor smooth muscles of the guinea-pig urinary bladder. J Physiol 2004;559: [27] Davidson RA, McCloskey KD. Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons. J Urol 2005;173: [28] Troxel SA, Jones AW, Magliola L, Benson JS. Physiologic effect of nifedipine and tamsulosin on contractility of distal ureter. J Endourol 2006;20: [29] Sergeant GP, Thornbury KD, McHale NG, Hollywood MA. Interstitial cells of Cajal in the urethra. J Cell Mol Med 2006;10: Editorial Comment on: Characteristics of Spontaneous Activity in the Bladder Trigone Christian Gratzke, Michael Seitz Department of Urology, Ludwig-Maximilians-University, Munich, Germany Christian.Gratzke@med.uni-muenchen.de The normal bladder functions storage and elimination of urine are based on a coordinated interplay of reciprocal contraction and relaxation of the bladder and the outflow region. This interaction is regulated by neural circuits in the brain and spinal cord that coordinate the activity of the detrusor and that of the smooth and striated muscles of the outflow region [1]. While NO/cGMPmediated relaxation of the trigone contributes to the inititation of the micturition cycle [2], the present study by Roosen et al [3] analyzes the role of the bladder trigone during bladder filling. The authors identify trigonal myocytes that show spontaneous activity in the form of [Ca2+]i as a consequence of transmembrane Ca2+ influx through L-type Ca2+ channels. Interestingly, the cellular origin of spontaneous activity in the trigone differs from other cells in the lower urinary tract such as detrusor myocytes and interstitial cells (ICs). Recent research has focused on the role of ICs in the bladder function [4]. A network of these cells with similar characteristics as the interstitial cells of Cajal that are involved in regulation of gastrointestinal motility is located beneath the urothelium in the human bladder and in the muscular wall of the detrusor. This network has been suggested to be involved in integrating signals from the urothelium, sensory nerves, and bladder wall. ICs have also been located in the urethra and are known to exhibit spontaneous firing activity. The close structural relation between ICs, smooth muscle, and nerves forms a basis for the involvement of these cells in the regulation of urethral tone and the continence mechanism. Recently, TRPA1-immunoreactivity was detected in interstitial cells that were evenly distributed in the muscular wall and specifically located on the boundaries of human urethral smooth muscle cell bundles [5]. Together with the finding that extensive gap junction coupling contributes to spontaneous contraction of the trigone, the study by Roosen et al provides valuable information on the physiological function of trigone myocytes. Future studies are needed to evaluate further the mechanisms leading to relaxation of the trigone in particular and the importance of interstitial cells in the regulation of bladder function in general. References [1] Andersson KE, Arner A. Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 2004;84:

9 354 EUROPEAN UROLOGY 56 (2009) [2] Persson K, Alm P, Johansson K, Larsson B, Andersson KE. Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry, and functional effects. Br J Pharmacol 1993;110: [3] Roosen A, Wu C, Sui G, et al. Characteristics of spontaneous activity in the bladder trigone. Eur Urol 2009;56: [4] Drake MJ. The integrative physiology of the bladder. Ann R Coll Surg Engl 2007;89: [5] Gratzke C, Streng T, Waldkirch E, et al. Transient Receptor Potential A1 (TRPA1) activity in the human urethra evidence for a functional role for TRPA1 in the outflow region. Eur Urol 2009; 55: DOI: /j.eururo DOI of original article: /j.eururo Editorial Comment on: Characteristics of Spontaneous Activity in the Bladder Trigone Petter Hedlund Department of Clinical Pharmacology, Lund University, Linköping University, Sweden petter.hedlund@med.lu.se Isolated preparations from the detrusor from various species, including humans, exhibit spontaneous contractile activity [1]. Similarly, autonomous nonmicturition contractions (NMCs) are observed under experimental conditions in isolated, whole rodent bladders and can also be recognized during cystometry in normal animals [2]. The underlying mechanisms of the spontaneous activity in vitro or in vivo are not established, and it is not known what the phenomena represent for bladder function. It is proposed that spontaneous activity may be of importance for functions such as sensory functions and pressure regulatory functions of the micturition reflex and that activity in the urothelium, nerves, interstitial cells, and smooth muscle are involved [1,2]. A role for detrusor overactivity (DO) may also be considered because increased spontaneous activity in vitro and in vivo of the bladder is reported in lower urinary tract (LUT) dysfunction [1]. Gap junctions (connexins) allow the spread of spontaneous-action potentials of bladder tissue from rodents and humans by coupling of cells [1]. In the present study, Roosen et al described a higher expression of connexin 43 in trigonal preparations than in the detrusor and also showed that trigonal spontaneous activity could be modified by blockade of gap junctions [3]. These results corroborate the findings that trigonal tissue exhibited a higher incidence of spontaneous activity than preparations from the dome. Roosen et al also show other similarities and differences in the cellular mechanism of trigonal activity compared to previous findings in detrusor myocytes and LUT interstitial cells [1,3]. Taking these findings together, the study supports a specialized function for the trigonal area during storage of urine. Of further interest are the findings that calcium channels and gap junctions are involved in trigonal spontaneous activity, since altered activities in these cellular signaling mechanims have been described for bladders from patients with DO [4,5]. References [1] Andersson KE, Arner A. Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 2004;84: [2] Gillespie JI. The autonomous bladder: a view of the origin of bladder overactivity and sensory urge. BJU Int 2004;93: [3] Roosen A, Wu C, Sui G, Chowdhury RA, Patel PM, Fry CH. Characteristics of spontaneous activity in the bladder trigone. Eur Urol 2009;56: [4] Haferkamp A, Mundhenk J, Bastian PJ, et al. Increased expression of connexin 43 in the overactive neurogenic detrusor. Eur Urol 2004;46: [5] Sui GP, Wu C, Severs N, Newgreen D, Fry CH. The association between T-type Ca2+ current and outward current in isolated human detrusor cells from stable and overactive bladders. BJU Int 2007;99: DOI: /j.eururo DOI of original article: /j.eururo