Effects of imatinib mesylate (Glivec) as a c-kit tyrosine kinase inhibitor in the guinea-pig urinary bladder

AIMS: In the gastrointestinal tract, slow wave activity in smooth muscle is generated by the interstitial cells of Cajal (ICC). Detrusor smooth muscle strips of most species show spontaneous contractions which are triggered by action potential bursts, however, the pacemaker mechanisms for the detrusor are still unknown. Recently, ICC-like cells have been found in guinea-pig bladder, using antibodies to the c-kit receptor. We have investigated the effects of Glivec, a c-kit tyrosine kinase inhibitor, on spontaneous action potentials in guinea-pig detrusor and intravesical pressure of isolated guinea-pig bladders. METHODS: Changes in the membrane potential were measured in guinea-pig detrusor smooth muscle using conventional microelectrode techniques. Pressure changes in the bladder were recorded using whole organ bath techniques. RESULTS: Smooth muscle cells in detrusor muscle bundles exhibited spontaneous action potentials, and spontaneous pressure rises occurred in isolated bladders. Glivec (10 microM) converted action potential bursts into continuous firing with no effects on the shape of individual action potentials. Glivec (>50 microM) reduced the amplitude of spontaneous pressure rises in the whole bladder in a dose dependent manner and abolished spontaneous action potentials in detrusor smooth muscle cells. CONCLUSIONS: The results suggest that ICC-like cells may be responsible for generating bursts of action potentials and contractions in detrusor smooth muscle. Drugs inhibiting the c-kit receptor may prove useful for treating the overactive bladder.     

大鼠膀胱ICC样细胞与逼尿肌神经调控关系的形态学研究

目的 从形态学上探讨膀胱ICC样细胞在逼尿肌神经调控中的作用.方法 采用透射电镜观察大鼠膀胱内ICC样细胞、神经和逼尿肌细胞之间的超微结构关系.通过c-kit免疫荧光染色对大鼠膀胱ICC样细胞进行鉴定,并通过c-kit与PGP9.5免疫荧光双标观察ICC样细胞与神经的结构关系.结果 透射电镜显示,ICC样细胞与逼尿肌细胞紧密相邻处可见典型缝隙连接.在局部区域可见ICC样细胞的突起与神经末梢联系紧密.c-kit免疫荧光染色显示,大鼠膀胱内ICC样细胞主要位于黏膜下层、肌束边缘以及肌细胞间.c-kit与PGP9.5免疫荧光双标显示ICC样细胞与神经末梢在结构上关系紧密.结论 从形态学上看,膀胱内ICC样细胞具备参与逼尿肌神经调控的结构基础,进一步从功能学上予以证实将有助于全面阐明逼尿肌神经调控理论.     

Efficacy of the beta3-adrenergic receptor agonist CL-316243 on experimental bladder hyperreflexia and detrusor instability in the rat

PURPOSE: Recent evidence indicates that in a number of species detrusor relaxation is mediated through activation of the beta3-adrenergic receptor. We determined whether activation of the beta3-adrenergic receptor would be a useful therapeutic approach for bladder instability. We profiled in vitro activity of the beta3-adrenergic receptor agonist CL-616243 and the efficacy of this compound in experimental models of detrusor instability and bladder hyperreflexia. MATERIALS AND METHODS: Isolated rat detrusor strips were contracted by depolarizing the preparations with 20 mM. KCl. CL-316423 was added to the tissue bath in increasing concentrations and contraction inhibition was assessed. Efficacy against bladder instability was evaluated using the obstructed hypertrophied bladder model in the rat. The acetic acid bladder cystometry model was used to assess the efficacy of CL-316423 in bladder hyperreflexia. Isovolumetric contractions were evoked by electrical stimulation using a silver bipolar electrode. Data are expressed as the mean plus or minus standard error of mean. RESULTS: CL-316243 inhibited spontaneously contracting, isolated rat detrusor strips in a concentration dependent manner with a mean concentration inhibiting 50% of maximal response of 2.65 +/- 0.36 nM. Intrinsic activity relative to forskolin was 1. In vivo CL-316243 administered intravenously or orally significantly increased the voiding interval and bladder compliance. In addition, there was a decrease in the number of spontaneous contractions during the filling phase in a model of neurogenic and obstruction induced bladder instability. The amplitude of electrically evoked isovolumetric contractions was significantly smaller after CL-316243 exposure. CONCLUSIONS: These data suggest that activating the beta3-adrenergic receptor in rat bladder using CL-316243 may directly inhibit smooth muscle contractility, experimental hyperreflexia and detrusor instability, and be useful for urge urinary incontinence.     

Depressed contractile responses to neurokinin A in idiopathic but not neurogenic overactive human detrusor muscle

OBJECTIVE: The role of tachykinins such as neurokinin A in regulating bladder function is unclear, but NK2 receptors seem to mediate contraction in the human bladder and it has been suggested that these peptides may have a role in the pathophysiology of bladder dysfunction. The present study investigates neurokinin receptor-mediated contractility of detrusor muscle in the idiopathic overactive and neurogenic overactive bladder and investigates the neurokinin receptor subtypes involved. METHODS: Human bladder was obtained from patients undergoing cystectomy (normal) or clam cystoplasty (idiopathic overactive) and from patients with spinal injuries (neurogenic overactive). Strips of isolated detrusor muscle were mounted in physiological Krebs-bicarbonate solution and cumulative concentration-response curves to 1 nM to 300 microM neurokinin A (NKA) were obtained in the absence and presence of neurokinin receptor antagonists, either the NK2 receptor-selective antagonist SR 48968 or the NK3 receptor-selective antagonist SB 223412. RESULTS: NKA evoked concentration-dependent contraction of normal, idiopathic, and neurogenic overactive detrusor strips. In idiopathic overactive detrusor muscle, NKA-induced contraction was significantly reduced relative to normal detrusor (0.031 +/- 0.005 mg/g, n = 11 versus 0.193 +/- 0.039 mg/g, n = 7). Sensitivity to the peptide was also significantly (p < 0.01) reduced in idiopathic overactive detrusor, with mean pEC50 values (concentration producing 50% maximal response) of 6.62+/-0.16 (n = 11) compared to 7.47+/-0.19 (n = 7) in normal detrusor. In contrast, NKA-induced responses of neurogenic overactive detrusor were similar to those in normal detrusor, with a mean maximum contraction of 0.199 +/- 0.036 mg/g (n = 10) and mean pEC50 value of 7.85+/-0.16 (n = 10). NKA curves in all groups were shifted to the right by the NK2 receptor-selective antagonist SR 48968 with high affinity, pK(B) values being similar in normal, idiopathic, and neurogenic overactive detrusor (8.85 + 0.08, n = 14; 8.97 +/- 0.13, n = 12; 8.73 +/- 0.12, n = 8, respectively). In contrast the NK3 receptor-selective antagonist SB 223412 had a minimal effect on NKA responses and affinity values were low (pK(B) 5.81 +/- 0.11, n = 12 in normal; 5.75 +/- 0.08, n = 12 in idiopathic overactive, and 5.77 +/- 0.13, n = 11 in neurogenic overactive). CONCLUSION: These data indicate that NKA-induced responses are impaired in detrusor muscle from idiopathic overactive human bladder, but not in detrusor muscle from neurogenic overactive bladder. The NK2 receptor subtype appears to mediate NKA responses in the normal, idiopathic overactive, and neurogenic overactive detrusor. This is important evidence suggesting a difference between the bladder pathophysiology observed in idiopathic versus neurogenic overactive detrusor.     

In vitro and in vivo relaxation of urinary bladder smooth muscle by the selective myosin II inhibitor,blebbistatin

What’s known on the subject? and What does the study add? Various pathological conditions of the bladder, including overactive bladder syndrome (OAB), are associated with unregulated increases in bladder smooth muscle (SM) contraction. Although a number of new pre‐clinical pharmacological agents for OAB have been identified, they predominantly target at the neural or detrusor cell membrane level rather than the SM contractile apparatus itself. The current study provides the first demonstration that blebbistatin, a novel small cell permeable molecule with demonstrated high affinity and selectivity toward the myosin II contractile molecule, potently and efficiently relaxes both rat and human bladder SM in vitro and also significantly alters urodynamic parameters in vivo to values reflective of decreased bladder overactivity. OBJECTIVE To investigate the in vitro and in vivo effects of blebbistatin (a small cell‐permeable molecule with high affinity and selectivity toward the myosin II contractile molecule) on bladder smooth muscle (SM) contractility, as antimuscarinic therapy is only 65–75% effective in treating overactive bladder (OAB) and is associated with considerable side‐effects, with a <25% continuation rate at 1 year. MATERIALS AND METHODS Bladder and aortic strips from adult male rats, and human bladder strips obtained from open prostatectomy, were used for organ‐bath studies of blebbistatin. Awake cystometry was also used in rats in both the presence and absence of intravesically delivered blebbistatin. RESULTS Blebbistatin dose‐dependently and completely relaxed both KCl‐ and carbachol‐induced rat detrusor and endothelin‐1‐induced human bladder contraction. Pre‐incubation with 10 µm blebbistatin attenuated carbachol responsiveness by ≈65% while blocking electrical field stimulation‐induced bladder contraction reaching 50% inhibition at 32 Hz. The basal tone and amplitude of spontaneous contraction were also significantly diminished. Urodynamic variables were obviously altered by intravesical infusion with blebbistatin. CONCLUSION Our novel data show that blebbistatin strongly relaxes both rat and human bladder contraction induced by various physiological stimuli. Coupled with our in vivo data showing that nanomole doses of blebbistatin significantly alter urodynamic variables to produce a less active bladder, our results suggest the possibility of intravesically administered blebbistatin binding at myosin II being developed as a therapeutic treatment for OAB via a novel targeting of the SM contractile apparatus.     

Effect of anoxia-glucopenia and re-superfusion on intrinsic nerves of mammalian detrusor smooth muscle: importance of glucose metabolism

AIMS: To investigate the effect of anoxia/glucopenia and re-superfusion on intrinsic nerves in the mammalian urinary bladder. METHODS: Strips of detrusor smooth muscle were dissected from monkey and human urinary bladder and mounted for tension recording in organ baths superfused with Krebs solution. Human, monkey, and guinea-pig urinary bladders were treated to evaluate glycogen contents by a biochemical method. RESULTS: Detrusor strips from both monkeys and humans had to be exposed to anoxia-glucopenia for up to 2-2.5 hr to observe a progressive decline in the response to electrical field stimulation (EFS) of the intrinsic nerves, at variance with guinea-pig detrusor strips. In contrast, the response to direct activation of the smooth muscle with carbachol remained almost unaltered. Incubation of human and monkey detrusor strips with 2-deoxyglucose (2-DG) during 1 hr anoxia-glucopenia, however, caused a marked damage to the intrinsic nerves. The glycogen contents of both human detrusor specimens and monkey urinary bladders were 2.0- and 1.4-fold higher, respectively, than that found in guinea-pig urinary bladder; furthermore, untreated monkey detrusor sections showed a greater number of glycogen granules as compared to those subjected to anoxia-glucopenia and re-superfusion. In guinea-pig and in monkey detrusor sections glycogen granules were found in smooth muscle cells but not in neurons of intramural ganglia. CONCLUSIONS: A higher susceptibility of guinea-pig as compared to monkey and human nerves has been demonstrated; it is suggested that anaerobic glucose metabolism during anoxia-glucopenia is crucial for the functional recovery of detrusor intrinsic nerves from damage caused by anoxia-glucopenia and re-superfusion.     

Loss of ryanodine receptor calcium-release channel expression associated with overactive urinary bladder smooth muscle contractions in a detrusor instability model

OBJECTIVE: To investigate the changes in spontaneous bladder smooth muscle contractions that occur during detrusor instability (DI), and to test the possibility that altered function or expression of ryanodine receptors (RyRs) could account for the increased bladder contractions. MATERIALS AND METHODS: After 8 weeks of partial bladder outlet obstruction, DI was confirmed in female experimental rats by filling cystometry. Muscle strips were dissected from freshly isolated bladders, and isometric tension recorded in strips from DI and normal bladders. The contractions were recorded during electrical stimulation or exposure to various agents. Western blot analysis was used to determine RyR expression in DI and normal bladder muscle. RESULTS: In DI bladder muscle, spontaneous contractile activity persisted in the presence of blockers for known neurotransmitter receptors in the bladder wall. The RyR blocker ryanodine significantly increased the spontaneous contractile frequency in normal bladder strips, but failed to affect spontaneous contractions in DI muscle. Caffeine inhibited spontaneous contractile activity in both the DI and normal strips. After administering the l-type Ca(2+) channel antagonist nimodipine, the myogenic contractile activity was abolished in normal strips; in contrast, in DI strips, the amplitude of contractions was reduced but the frequency of contractions was unchanged. Western blot analysis showed that RyR expression was lower in DI muscle than in normal bladder muscle. CONCLUSION: These results provide the first characterization of a loss of regulation of spontaneous contractile activity by RyRs in DI muscle associated with a significant decrease in RyR expression. RyRs in normal detrusor muscle act as negative-feedback regulators of spontaneous contractile activity, presumably by releasing Ca(2+) that activates Ca(2+)-dependent K(+) channels to decrease contractility. This mechanism might be weakened in DI muscle, resulting in spontaneous contractile overactivity.     

The elusive electromyogram in the overactive bladder: a spark of understanding

It has been said that a technique capable of recording a urinary bladder electromyogram could be useful in the clinical evaluation of the detrusor neuropathies and myopathies implicated in the generation of lower urinary tract symptoms. However, in contrast to electromyography of skeletal and cardiac muscle, detrusor smooth muscle electromyography has remained in its infancy despite 50 years of scientific effort. The principal problems appear to be isolation of the real signal from artefacts, and the doubtful existence of electromyographic activity during cholinergic muscle contraction. The discovery of purinergic neuromuscular transmission in the overactive human bladder has renewed interest in detrusor electromyography as, in contrast to cholinergic mechanisms, purinergic mechanisms can generate extracellular electrical activity. In this paper, the development and validation of a novel technique for recording electrical activity from neurologically intact guinea-pig and human detrusor in vitro is described. A purinergic electromyographic signal is characterised and it is shown that detrusor taken from overactive human bladders has a greater propensity to generate electromyographic activity than normal by virtue of an aberrant purinergic mechanism.     

Bladder cooling reflex in patients with multiple sclerosis

PURPOSE: We describe the effect of intravesical ice water instillation in patients with multiple sclerosis and without an overactive bladder. MATERIALS AND METHODS: Of 131 consecutive patients with multiple sclerosis who presented with a urinary disorder we selected for study 10 men and 29 women with a mean age plus or minus standard deviation of 50 +/- 9 years who had multiple sclerosis without an overactive bladder. Nonoveractive bladder was defined as no involuntary detrusor contraction up to 400 ml. of maximum fill on routine cystometry. We performed cystometry with saline at 25 to 30C at an infusion rate of 50 and 100 ml. per minute, and with ice water at 0 to 4C at a rate of 100 ml. per minute. Ice water cystometry was considered positive when an involuntary detrusor contraction occurred before 200, and between 200 and 400 ml. of filling. Ice water cystometry was considered negative when there was no involuntary detrusor contraction during ice water filling up to 400 ml. RESULTS: Ice water cystometry enabled us to elicit involuntary detrusor contractions in 21 patients, which remained undetected by warm water cystometry at rates of 50 and 100 ml. per minute. The test was positive before 200, and between 200 and 400 ml. in 10 and 11 cases, respectively. Positive ice water cystometry was significantly associated with irritative signs or significant post-void residual urine volume. CONCLUSIONS: An involuntary detrusor contraction was not elicited by cystometry at 50 or 100 ml. per minute, implying that the afferent mechanoreceptor reflex limb via ADelta fibers is not involved. In contrast, ice water cystometry at 100 ml. per minute elicited an involuntary detrusor contraction, suggesting involvement of an afferent reflex limb via capsaicin sensitive C fibers. These involuntary detrusor contractions revealed by ice water cystometry are probably relevant to an overactive bladder. In urinary disorders such a positive test indicates a spinal lesion. In multiple sclerosis it may have pathophysiological value, indicating a spinal rather than cerebral mechanism of overactive bladder, and diagnostic value, indicating multifocal demyelination.     

Prostaglandin type E activity dominates in urinary tract smooth muscle in vitro

Changes in isometric tension and spontaneous contractions in human and porcine lower urinary tract smooth muscle strips caused by PGE2, PGF2a and the prostaglandin biosynthesis inhibitor ketoprofen were investigated in vitro. Ketoprofen reduced the prostaglandin biosynthesis significantly and caused reversible and dose dependent decreases of tension and spontaneous contractions in detrusor strips, while tension increase was induced in strips from the urethra, bladder neck and trigone. PGE2 reestablished the tension and the spontaneous activity in the detrusor strips and counteracted the tension increase in strips from the urethra, bladder neck and trigone. PGF2a increased the tension in all strips. The threshold concentration for prostaglandin effect in strips pretreated with ketoprofen was 10(-10) - 10(-9) mol./l., a concentration which might be considered physiologically relevant. Prostaglandin synthesis inhibition thus caused responses opposite to those of PGE2 in every single strip. These results strongly suggest the PGE-type of activity to be more important than the PGF-type of activity in lower urinary tract smooth muscle in vitro.     

The effects of a new selective beta3-adrenoceptor agonist (GW427353) on spontaneous activity and detrusor relaxation in human bladder

OBJECTIVE: To examine the effects of a new selective beta3-adrenoceptor agonist, GW427353 on human detrusor function, as beta2- and beta3-adrenoceptors have been identified in the bladder, and can mediate detrusor relaxation, but beta3-adrenoceptors are less widely distributed and beta3-adrenoceptor agonists should have the therapeutic advantage of producing fewer treatment side-effects. PATIENTS AND METHODS: 'Normal' human detrusor was retrieved from 12 patients (mean age 56 years) at cystectomy and from organ donors. Detrusor strips (4 x 1 x 1 mm) were mounted in superfused organ baths. Tone was induced with carbachol (5 x 10(-7)m) before applying either a nonselective beta-adrenoceptor agonist (isoprenaline) or GW427353 (with or without the beta3-adrenoceptor antagonist, SR59230A). In addition, the effect of GW427353 was tested on intrinsic nerve-evoked smooth muscle contraction over time. Effects on spontaneous activity were also recorded. RESULTS: GW427353 produced significant relaxation at concentrations of >10(-7)m; isoprenaline produced a significant effect from 10(-6)m, but otherwise both agonists had similar effects. The addition of SR59230A (10(-7)m), produced partial inhibition of the GW427353 response. GW427353 at 10(-6)m significantly reduced spontaneous activity within 10 min of incubation, and at higher concentrations (>5 x 10(-6)m) inhibited detrusor contractions evoked by electrical field stimulation. CONCLUSION: Neuropathic bladder dysfunction is characterized by increased spontaneous activity and involuntary detrusor contractions, which can result in urinary frequency, urgency, nocturia and incontinence. The novel feature of GW427353 is the ability to suppress spontaneous activity and produce significant relaxation in human detrusor tissue at low concentrations, whilst also inhibiting evoked detrusor contractions at higher concentrations.     

Inhibitory effect of a calcium entry blocker (verapamil) on detrusor muscle contractility: feasibility of clinical application

Effects of a Ca2+ entry blocker (verapamil) on the contractility of the bladder detrusor muscle of rabbits were investigated in vitro and in vivo. In experiments using smooth muscle strips from the bladder detrusor, isometric tension changes of the strips following drug addition were recorded. The contractions of the strips induced by acetylcholine (10(-8)-10(-2)M), prostaglandin E2, F2-alpha (3 X 10(-8)-3 X 10(-5)M) or electric stimulation were significantly inhibited dose-dependently by pretreatment of the strips with verapamil (10(-7), 10(-6)M). In in vitro experiments using whole bladder preparations, the spontaneous contractile activity and the contraction induced by acetylcholine (10(-6)M) were monitored. Both activities were inhibited in a time-dependent manner after the intravesical instillation of 7.5 mg verapamil. The amplitude of the spontaneous contraction and responses to acetylcholine, 90 minutes after the instillation, were reduced to 10% and 38% of the control levels (before the instillation), respectively. The detrusor contractility was still inhibited 2 hours after the removal of verapamil from the bladder. During in vivo experiments, changes in intravesical pressure and systemic arterial pressure were monitored. Sixty minutes after the intravesical instillation of 10 mg verapamil, the rise of the intravesical pressure following the pelvic nerve stimulation was inhibited to 18% of the control level, whereas the systemic arterial pressure was not affected. Verapamil is suggested to have potent inhibitory effects on the detrusor muscle contraction, and the intravesical instillation of verapamil to inhibit detrusor contractility without affecting the cardiovascular status.     

The inhibitory role of melatonin on isolated guinea-pig urinary bladder: an endogenous hormone effect

OBJECTIVE: To investigate the effects of melatonin, an endogenous hormone, on acetylcholine and KCl-induced contractions of isolated guinea-pig detrusor muscle. MATERIALS AND METHODS: Detrusor smooth muscle strips isolated from guinea-pig bladders were placed in an organ bath containing physiological saline at 37 degrees C and pH 7.4, constantly bubbled with 95% oxygen and 5% CO2. The effects of cumulatively applied melatonin on the acetylcholine- and KCl-induced contractions of isolated bladder strips were examined using isometric contraction measurements. RESULTS: Melatonin (100 and 300 micromol/L) significantly inhibited the peak amplitude of both acetylcholine (10 micromol/L) and KCl (30 mmol/L)-induced contraction of the isolated bladder strips (P < 0.05). Similarly, melatonin caused a significant reduction in the contractile frequency induced by KCl (eight strips) in a concentration-dependent manner, while having no significant effect on the frequency of contractile response to acetylcholine, even at the highest concentration (300 micromol/L) used (P = 0.58, 14 strips). CONCLUSIONS: These results suggest that melatonin inhibits acetylcholine- and KCl-induced contractions in isolated bladder strips from guinea pigs. The endogenous nature of melatonin, with its low side-effect profile, makes it a potentially useful agent to be considered in the medical management of the overactive bladder.     

前列冲剂对雄性大鼠离体膀胱逼尿肌作用的研究

目的观察前列冲剂对雄性大鼠离体膀胱逼尿肌运动的影响，并初步探讨其作用机制。方法运用BL-420型生物信号系统记录离体大鼠膀胱逼尿肌肌条的活动，观察不同剂量前列冲剂对大鼠膀胱逼尿肌自发活动的影响，并分析其作用机制。结果前列冲剂对大鼠膀胱逼尿肌有明显的兴奋作用，可增加其收缩时间、最大收缩张力及膀胱活动力，并呈量效关系。该作用可被异搏定（L型电压依从性Ca2＋通道阻断剂）和阿托品部分阻断（P〈0.01）。结论前列冲剂在体外可增强膀胱平滑肌的收缩，其作用是通过多条途径发挥的，尤其是通过L型钙通道和胆碱能M型受体而起作用的。     

Cajal-like cells in the human upper urinary tract

PURPOSE: Interstitial cells of Cajal (ICCs) have an important role in the regulation of gut motility as they are responsible for the slow wave activity of smooth muscle. It is still unknown if ICCs also occur in the human upper urinary tract. Since these cells express and are marked by the c-kit receptor CD117, we investigated its occurrence and distribution along the human upper urinary tract. MATERIALS AND METHODS: Tissues from 56 human ureters, spanning proximal, middle and distal ureter segments, were analyzed by indirect immunohistochemistry using the alkaline phosphatase-anti-alkaline phosphatase method and double labeling immunofluorescence on consecutive tissue sections. Several monoclonal and polyclonal antibodies to c-kit receptor were used in combination with various cell markers for histiocytic, mast cell, endothelial, epithelial, neuronal, smooth muscle and stem cell differentiation. RESULTS: The c-kit receptor was found in 3 cell types of the ureter and in round or spindle-shaped cells. Due to their antigenic profile the first one was revealed as mast cells occurring in all layers of the ureteral wall except the urothelium. In contrast, the population of spindle-shaped cells was only marked by c-kit receptor, thus, resembling ICCs. These ICC-like cells were found among the inner and outer smooth muscle layers, and in the lamina propria. They showed a slight decrease from proximal to distal ureteral segments. However, unlike intestinal ICCs their cytomorphology differed and some cells, representing the third group of c-kit receptor positive cells, were found within the urothelium. CONCLUSIONS: Our data demonstrate the presence of ICC-like cells and their ubiquitous distribution in the human ureter. The physiological importance and pathological significance of these findings must be evaluated by functional studies and investigations of certain pathological with urinary outflow disturbance conditions.     

Spontaneous activity of mouse detrusor smooth muscle and the effects of the urothelium

AIMS: To characterize the detrusor muscle of the mouse urinary bladder in order to understand more precisely spontaneous contractile behavior of this organ. This study examined the spontaneous electrical activity and Ca(2+) dynamics of the detrusor smooth muscle and investigated the role of the urothelium. MATERIALS AND METHODS: Detrusor smooth muscle strips were isolated from mouse bladders. The urothelium was either kept intact or removed. Changes in membrane potential were recorded using sharp electrode intracellular recording. To image Ca(2+) dynamics, tissue strips were exposed to 10 microM Oregon Green 488 BAPTA-1 AM for 70 min, and then image series were acquired with a laser-scanning confocal microscope. RESULTS: (1) Mouse detrusor smooth muscle cells (SMCs) generate nifedipine-sensitive spontaneous action potentials (sAPs) at a low frequency (1.3 +/- 0.9 min(-1), n = 11) in preparations with intact urothelium. This frequency increased when the urothelium was removed (7 +/- 8.3 min(-1), n = 17) (P < 0.05, Student's t test). (2) Frequent ATP-mediated spontaneous depolarizations were recorded in all cells. (3) The frequency of whole cell Ca(2+) flashes of detrusor smooth muscle cells was higher in preparations with the urothelium removed (median 1.2 min(-1), n = 7) than in urothelium denuded preparations (median 0.6 min(-1), n = 7) (P < 0.01, Mann-Whitney U-test). CONCLUSIONS: Spontaneous activity of the mouse detrusor smooth muscles was characterized enabling future comparative work on gene knock-out strains. Evidence suggesting release of an inhibitory factor by the urothelium was apparent.     

Cystometric and in vitro muscle studies of cystoplastic appendiceal segments in the rat

AIMS: The functional integration of the smooth muscle of enterocystoplasties into the detrusor muscle was investigated in an awake-rat cystometry model and in vitro. METHODS: The upper fourth of the bladder was removed, and a detubularized appendiceal segment (7 x 7 mm), with preserved vasculature, was incorporated into the bladder. After 1 or 3 months, a catheter was fixed to the top of the bladders. After a 3-day recovery, cystometries were performed. In separate experiments, agonist and nerve-induced responses were evaluated on isolated bladder strips. RESULTS: Cystometries revealed reduced basal pressure and micturition pressure in enterocystoplasty (ECP) bladders. Bladder capacity and micturition volume were increased. Threshold pressure (pressure immediately before micturition) was significantly lower at 1 month, but not at 3 months. Bladder compliance was significantly higher in the operated at 1 month but not at 3 months. Threshold tension did not differ between control and corresponding operated groups. Residual urine was significantly higher in the operated groups. ECP strips showed increased maximal contractions to nerve stimulation, to levels similar to those of detrusor strips. Maximal responses to carbachol increased to levels between those of intestine and detrusor. The inhibitory effect of scopolamine on nerve induced contractions increased to levels similar to those for detrusor. Purinergic activation had no effect on intestinal or ECP strips, but contracted detrusor muscle. CONCLUSIONS: The smooth muscle of the bowel segment in rat ECP bladders underwent a partial change in the response to nerve stimulation from that of intestine towards that of detrusor. The cystometry experiments suggested a partial functional integration of the ECP segment into the detrusor.     

Vasoactive intestinal polypeptide influence on lower urinary tract smooth muscle from human and pig

The influence of vasoactive intestinal polypeptide on detrusor, trigone, bladder neck and urethral smooth muscle from human and pig was investigated in vitro. Vasoactive intestinal polypeptide reduced the tension and amplitude of the spontaneous contractions of strips from all regions studied. Human detrusor and pig trigone, bladder neck and urethral strips were more sensitive to vasoactive intestinal polypeptide than pig detrusor. The response was reversible, reproducible and dose-dependent from 10(-9) to 10(-6) mol. per liter. The time to onset of the response was within 1/2 minute and the time to maximal relaxation was 2 to 10 minutes. The response was not affected by selective nerve poisoning with tetrodotoxin, beta-adrenergic blockade with propanolol or prostaglandin synthesis blockade with ketoprofen. Vasoactive intestinal polypeptide did not prevent prostaglandin E2 activity on the musculature. Contractions evoked by transmural electric field stimulation or pharmacologically with carbachol, noradrenaline, substance P and prostaglandin F2 alpha were equally reduced by VIP 10(-7) mol. per liter.     

Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons

PURPOSE: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells. MATERIALS AND METHODS: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy. RESULTS: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive. CONCLUSIONS: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.     

The role of Ni(2+)-sensitive T-type Ca(2+) channels in the regulation of spontaneous excitation in detrusor smooth muscles of the guinea-pig bladder

OBJECTIVE: To explore the role of Ni(2+)-sensitive T-type Ca(2+) channels in the generation of spontaneous excitation of detrusor smooth muscles. MATERIALS AND METHODS: In isolated detrusor smooth muscle bundles of the guinea-pig bladder, changes in the membrane potential and muscle tension were measured using intracellular microelectrodes and isometric tension recording. Changes in the intracellular Ca(2+) concentration were recorded from bundles loaded with the fluorescent dye fura-PE3. RESULTS: Detrusor smooth muscles had two types of spontaneous electrical activity, i.e. individual and bursting action potentials. Ni(2+) (30 microM), a blocker for T-type Ca(2+) channels, reduced the frequency of individual action potentials without changing their amplitude. Higher concentrations of Ni(2+) (100-300 microM) converted individual action potentials into the bursts, as did apamin (0.1 microM), a blocker of small-conductance Ca(2+)-activated K(+) channels (SK). They also increased the amplitudes of spontaneous Ca(2+) transients and corresponding contractions whilst reducing their frequencies. In preparations which generated bursting action potentials, nifedipine (1 microm) converted action potentials into spontaneous transient depolarizations (STDs), and subsequent applications of Ni(2+) (100 microm) abolished STDs. Gadolinium (100 microM) and SKF96365 (10 microM), blockers for nonselective cation channels, and niflumic acid (100 microm), a blocker for Ca(2+)-activated Cl- channels, had no effect on either the amplitude or frequency of spontaneous action potentials. CONCLUSIONS: The T-type Ca(2+) channel may have dual roles in generating spontaneous excitation in detrusor smooth muscles. First, activity of these channels may account for the preceding depolarizations that lead to action potentials. Second, Ca(2+) influx through T-type Ca(2+) channels may couple functionally to SK channels, contributing to the stability of the resting membrane potential in detrusor smooth muscle. Thus, pharmacological manipulation of T-type Ca(2+) channels in detrusor smooth muscles could be of potential value for treating the overactive bladder.