Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction

AIMS: We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. METHODS: Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. RESULTS: Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. CONCLUSIONS: These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.     

Characterization of outward currents in interstitial cells from the guinea pig bladder

PURPOSE: Outward currents were characterized from cells resembling interstitial cells of Cajal (ICCs) isolated from the detrusor of the guinea pig bladder. MATERIALS AND METHODS: ICC-like cells were studied using the whole cell patch clamp technique and K+ filled pipettes. Outward currents were evoked by stepping positively from a holding potential of -80 mV. RESULTS: ICC-like cells were distinguished from smooth muscle cells by the presence of lateral branches and an inability to contract spontaneously or when depolarized. Depolarization elicited large outward currents. Penitrem A, a blocker of large conductance, Ca activated K+ channels, significantly decreased the outward current. Its Ca dependence was demonstrated by significant inhibition with nifedipine and Ca-free solution. When large conductance, Ca activated K+ and Ca currents were blocked with penitrem A and nifedipine, a voltage dependent current was unmasked, which activated positive to -50 mV and displayed voltage dependent inactivation with half-maximal inactivation occurring at -71 mV. It was blocked in concentration dependent fashion by tetraethylammonium but unaffected by 4-aminopyridine, charybdotoxin or apamin, suggesting that small and intermediate conductance, calcium activated potassium channels, and Kv1.2 and Kv1.3 channels are unlikely to be involved. At maximal concentrations of tetraethylammonium a portion of the voltage dependent K+ current remained that was not affected by any of the blockers tested. CONCLUSIONS: ICC-like cells from the detrusor possess calcium activated and voltage dependent K+ currents.     

Cajal样间质细胞在豚鼠膀胱肌层的分布及意义

目的研究豚鼠膀胱Cajal样间质细胞的分布、超微结构特点并探讨其功能。方法选择健康豚鼠10只,利用激光共聚焦显微镜和透射电镜观察膀胱Cajal样间质细胞在肌层的分布和结构特点。结果豚鼠膀胱肌层Cajal样间质细胞形成网状和平行状两种不同的分布。超微结构核大、具有丰富的细胞器。结论膀胱Cajal样间质细胞分布和超微结构与胃肠道的ICCs相似,具有起搏细胞特征。     

机械牵张对豚鼠膀胱组织Cajal间质细胞形态学及兴奋性的影响

目的 探讨机械牵张对豚鼠膀胱组织Cajal间质细胞(ICC)形态及兴奋性的影响.方法 建立雌性豚鼠膀胱颈部分梗阻(PBOO)模型作为实验组.并设假手术组作为对照.术后4周取膀胱组织制片,免疫荧光染色观察2组ICC形态和分布情况;胶原酶消化豚鼠膀胱制细胞悬液,免疫荧光标记,流式细胞仪检测2组c-kit阳性细胞比率.在弹性硅胶膜七原代培养膀胱ICC,利用Fluo-4钙荧光指示剂检测机械牵张对ICC钙信号的影响.结果 2组豚鼠膀胱组织铺片均可见c-kit染色阳性典型长梭形有突起的ICC,主要分布于平滑肌肌束间;PBOO组膀胱平滑肌间质增厚,c-kit染色阳性细胞数量及其突起明显增多,互相连接呈网络状;PBOO组c-kit阳性细胞比率为(6.7±1.7)%,显著高于对照组的(1.0±0.5)%,差异有统计学意义(P＜0.05).体外培养的ICC可检测到自发性钙波,机械牵张刺激可诱导ICC发生钙波增强现象.结论 机械牵张可诱导膀胱ICC兴奋性增强;PBOO膀胱组织中ICC数量及相互联系显著增多.ICC可能参与了膀胱牵张感受功能并在长期牵张应力负荷下发生一定的适应性改变.     

豚鼠膀胱Cajal样间质细胞的分离、培养及其鉴定

目的探索豚鼠膀胱Cajal样间质细胞的原代培养方法。方法颈部脱臼法处死豚鼠，无菌条件下分离出膀胱肌组织制备成肌条，采用V型胶原酶酶解法分离细胞，将细胞悬液接种于含干细胞因子的细胞培养基中培养，隔日换液。用酪氨酸蛋白激酶受体特异性抗体标记细胞，证实细胞类型。结果培养后的Cajal样间质细胞保持其固有特征，可见胞体为梭形且胞体两极有两个细长突起，该类细胞酪氨酸蛋白激酶受体抗体荧光染色阳性证实细胞培养成功。结论用V型胶原酶消化法可获得成年豚鼠膀胱Cajal样间质细胞，并在体外条件下生长，可用于Cajal样间质细胞的电生理学研究。     

Noradrenaline inhibits autonomous activity in the isolated guinea pig bladder

OBJECTIVE: To investigate the actions of noradrenaline and the specific alpha-adrenergic agonists cirazoline (alpha1) and UK14304 (alpha2), and beta-receptor agonists formoterol (beta2) and BRL37344 (beta3) on the phasic activity induced by muscarinic stimulation on the isolated guinea pig bladder, as the physiological significance of this activity is unknown but it may underlie non-micturition contractions (NMCs, which can be inhibited by sympathetic nerve stimulation) and the generation of bladder sensations. MATERIALS AND METHODS: All experiments were conducted using whole isolated bladders from female guinea pigs (270-300 g). Bladders were cannulated via the urethra and suspended in a heated chamber containing oxygenated Tyrode's solution at 33-35 degrees C and the intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface. RESULTS: Exposure to noradrenaline reduced the amplitude and frequency of the phasic activity. When noradrenaline was washed off there was a transient increase in frequency. There was marked desensitization with repeated applications of noradrenaline. Applying the specific beta3-agonist BRL37344 reduced the amplitude of the phasic activity while formoterol, a specific beta2-agonist, had no effect. Cirazoline, a specific alpha1-agonist, reduced the amplitude of the responses and significantly reduced the frequency of the phasic activity. UK14304, a specific alpha2-agonist, had no effect. Stimulation of the hypogastric nerve to the guinea pig bladder generates contractions. Prolonged nerve stimulation at low frequency (6.5 Hz) generated phasic rises in intravesical pressure which were inhibited by noradrenaline. Using short (5 s) periods of stimulation noradrenaline inhibited nerve-mediated contractions at all frequencies but was more effective at <10 Hz. CONCLUSION: These experiments show that sympathomimetic stimulation in the isolated whole bladder results primarily in an inhibition of phasic activity, but also a stimulation. Two receptor subtypes appear to be involved in the inhibition, alpha1 and beta3, suggesting that there may be many sites of action. These results are discussed in terms of the possible physiological significance of phasic activity and the potential importance of its inhibition, in the context of the causes of pathological changes in the bladder, particularly those associated with bladder overactivity, and the pharmacological approach to the alleviation of clinical symptoms.     

豚鼠Oddi括约肌内Cajal样细胞及NOS表达阳性神经元的分布

目的研究Cajal样细胞和一氧化氮合酶(nitric oxide synthase,NOS)表达阳性神经元在成年豚鼠Oddi括约肌(sphincter of Oddi,SO)的分布。方法成年豚鼠SO冷冻切片,c-Kit免疫细胞化学和NADPH-黄递酶组织化学双重染色。结果SO横切面环行平滑肌层内可见少量c-Kit阳性细胞,胞体呈梭形,两端伸出细长的突起。纵切面SO的两个壁分别为:外侧壁和十二指肠壁内SO壁,前者与肠壁结构类似,在深肌丛和肌间神经丛周围可见较多的c-Kit阳性细胞;而十二指肠壁内SO壁的肌层内存在大量c-Kit阳性细胞和突起,其形态与肠壁肌层内Cajal细胞相似。NADPH-黄递酶组织化学染色可见NOS表达阳性神经元广泛分布于SO的肌间神经丛和平滑肌内。c-Kit免疫细胞化学和NADPH-黄递酶组织化学双重染色虽未发现二者的共存,但可见c-Kit阳性细胞及突起存在于NOS表达阳性神经元的附近。结论豚鼠SO内存在的Cajal样细胞可能参与SO自主节律性运动的调控,可能是NOS表达阳性神经元对SO运动发挥调节作用的靶细胞。     

豚鼠膀胱组织内Cajal样间质细胞及NOS表达阳性神经元的分布

目的探讨Cajal样间质细胞和一氧化氮合酶2（NOS2）表达阳性神经元在成年豚鼠膀胱组织内的分布。方法成年豚鼠膀胱冰冻切片,c—Kit免疫荧光和n—NOS2免疫荧光染色。结果膀胱肌层内可见c—Kit表达阳性细胞的存在,n-NOS2免疫荧光染色显示其阳性神经元广泛分布于膀胱组织内。c—Kit免疫荧光和n-NOS2免疫荧光双重染色虽未发现两者的共存,但可见c—Kit表达阳性细胞存在于NOS表达阳性神经元的附近。结论豚鼠膀胱的Cajal样间质细胞可能参与膀胱自主节律性运动的调控,提示二者可能共同参与膀胱自主节律性运动的调控,为“神经-Cajal样问质细胞-肌肉”作用单元的形成提供结构基础。     

Contractility of the guinea pig bladder measured in situ and in vitro

To study the relative importance of neurogenic factors in detrusor contractility and to relate a total bladder in vitro contractility model to a previously described bladder wall strip model, active intravesical pressure values were compared in situ and in vitro in eight male guinea pigs. In situ, the active pressure was measured in spontaneous isometric and nonisometric micturition contractions. In vitro, the active pressure was measured in isometric contractions of the same bladders, developed in response to optimal electrical stimulation. The volume dependence of the active pressure generated by the bladder was measured in vitro in order to relate bladder capacity to the volume where the generated force is maximal and to determine the optimal volume at which to study detrusor contractility. The results indicated that in normal micturition the detrusor muscle was not fully stimulated: active pressure in isometric contractions in vivo was about 60% of the pressure values attained in vitro at the same bladder volume. Most micturitions occurred at a volume where the active pressure generated in vitro was about 80% of the maximal pressure. The active pressure-bladder volume relationship complied with the sliding filament-cross bridge theory. In whole bladder preparations active stress was about twice as high as in strips. © 1994 Wiley-Liss, Inc.     

The action potential of guinea pig bladder smooth muscle

The smooth muscle of the guinea pig bladder demonstrates in vitro spontaneous electrical activity in the form of action potentials which are associated with contraction. The action potential frequency is highly voltage-sensitive. The relative contributions of Na, Ca and K to the action potential elicited by depolarizing current have been studied using intracellular microelectrodes. In solutions in which NaCl is replaced by sucrose, the membrane hyperpolarizes and the rate of rise and after-hyperpolarization of the elicited action potential is increased. The amplitude is unaffected. In Ca-deficient solutions, the membrane depolarizes, the rate of rise and amplitude of the action potential is reduced, and the after-hyperpolarization is decreased. Nifedipine reduces amplitude and rate of rise but does not affect after-hyperpolarization. In the presence of the K-channel antagonist TEA, the duration of the action potential is prolonged, but the amplitude and rate of rise are unaffected. After-hyperpolarization is not reduced. It is concluded that the action potential of guinea pig bladder muscle, like many other smooth muscles studied, is Ca-based. Repolarization depends on changes in K conductance. The after-hyperpolarization is voltage-sensitive.     

Electrical membrane events underlying contraction of guinea pig bladder muscle

The relationship of electrical to mechanical activity of guinea pig bladder muscle has been studied in vitro by intracellular microelectrode and sucrose gap techniques. A discrete relationship between individual electrical action potentials and muscle contractions of small amplitude can be seen in muscle which is spontaneously active or stimulated by muscarinic activation or electrical current. An increase in the frequency of action potentials is associated with an increase in the frequency of small-amplitude contractions and the generation of muscle tension. The frequency of action potential firing is sensitive to changes in the resting membrane potential of the muscle. The findings suggest that an increase in the frequency of action potential firing may be a major mechanism in the development of muscular tension in the wall of the bladder.     

Receptor operated intracellular calcium stores in the smooth muscle of the guinea pig bladder

Study of the in vitro behavior of strips of guinea pig bladder and taenia coli demonstrated that: Both bladder and taenia temporarily retain their ability to contract in Ca-free solutions, but the magnitude of this response decays with time. Carbachol is capable of producing contraction in Ca-free solution for a longer period of time than K depolarisation. Once lost, the ability of carbachol (but not K) to contract the tissues in Ca-free solution can be temporarily restored by a brief application of high Ca. The size of the carbachol contraction in Ca-free solution is reduced in Na-free solution, suggesting that membrane-bound Ca may not play a major role in this response. In depolarised bladder exposed to nifedipine 2 X 10(-7) M, carbachol can only elicit 1 large contraction, suggesting depletion of an intracellular source. It is concluded that whereas the response of the bladder to depolarisation depends primarily on extracellular Ca, the response to carbachol may also involve release of stored Ca and that the bladder, like other smooth muscles, appears to contain agonist-releasable intracellular Ca stores.     

Modulation of autonomous contractile activity in the isolated whole bladder of the guinea pig

OBJECTIVE: To investigate the actions of the nonhydrolysable analogue of ATP, alpha,beta-methylene ATP (alpha,beta-MATP) and the sensory peptide, substance P, on the phasic activity generated by muscarinic stimulation in the isolated whole bladder. Isolated bladder can generate complex contractions resulting in phasic rises in intravesical pressure (the autonomous bladder): activity thought to underlie nonmicturition activity in vivo and which may be important in generating bladder sensations. MATERIALS AND METHODS: Experiments were conducted on whole isolated bladders from female guinea pigs (270-300 g). Bladders were cannulated via the urethra, suspended in a heated chamber containing oxygenated solution at 33-36 degrees C and intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface of the bladder. RESULTS: When alpha,beta-MATP (30-3000 nmol/L) or substance P (30-300 nmol/L) was added to resting bladders there were small rises in intravesical pressure (<2 cmH2O). However, in the presence of phasic activity generated by exposing the bladder to the muscarinic agonist arecaidine (100-300 nmol/L) or the nicotinic ligand lobeline (10-30 micromol/L) similar or lower concentrations of alpha,beta-MATP or substance P produced more dramatic effects: alpha,beta-MATP and substance P (both at 100 nmol/L) activated a rise in basal pressure of > 15 cmH2O and increased the frequency of the phasic activity. On removing alpha,beta-MATP or substance P, there was a slowing of phasic activity indicative of an inhibitory mechanism. CONCLUSION: In addition to direct effects on smooth muscle the agonists alpha,beta-MATP and substance P appear to be potent regulators of the mechanisms generating phasic activity. A developing concept is that the mechanisms responsible for generating phasic activity underlie nonmicturition activity are the target for excitatory and inhibitory inputs. Regulating such activity may be a factor in generating or modifying bladder sensation. Inappropriate or exaggerated phasic activity could underpin the pathological changes which cause the overactive bladder, thus adding another hypothesis to the neurogenic and myogenic hypotheses of bladder overactivity, i.e. that of the autonomous bladder.     

Mitosis in normal adult guinea pig Leydig cells

In this study, Leydig cells in mitosis in adult guinea pigs were quantified. Testes of adult control guinea pigs (n = 10) were fixed by whole body perfusion with 2.5% glutaraldehyde in cacodylate buffer, postfixed in a mixture of osmium tetroxide-potassium ferrocyanide, and embedded in Epon Araldite for qualitative and quantitative microscopy. Using stereologic techniques, the total number of Leydig cells per testis and the number of dividing Leydig cells per testis were quantified. Light microscopic studies revealed the presence of dividing Leydig cells. Ultrastructural studies on a few of these Leydig cells showed that they contained abundant smooth endoplasmic reticulum and mitochondria, which further supported this identity. The total number of Leydig cells per testis and the number of dividing Leydig cells per testis were determined as 14.1 x 10(6) (standard error [SE] = 0.33) and 8 x 10(3) (SE = 5), respectively. These results indicate that Leydig cells undergo mitosis at a rate of 1 per 1.75 x 10(4) in adult guinea pigs.     

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.     

Changes in action potential kinetics following experimental bladder outflow obstruction in the guinea pig

Summary The effect of experimental bladder outflow obstruction on membrane electrical activity of guinea pig detrusor smooth muscle was studied. Using an intracellular microelectrode technique, action potentials were recorded from single smooth muscle cells to determine the effect of outflow obstruction on action potential (AP) kinetics. Bladder outflow obstruction resulted in smooth muscle hypertrophy with bladder weight gain to 2.7 times control levels after 8–12 weeks' obstruction. The changes in the AP kinetics noted with obstruction-induced bladder hypertrophy were a prolongation of the AP duration and a decrease in the maximum velocity of depolarization and repolarization. The AP amplitude, after hyperpolarization and overshoot potential in addition to the resting membrane potential (RMP) did not change significantly with bladder outflow obstruction. The values of these AP parameters were not affected significantly by the application of atropine and guanethidine in smooth muscle tissue from either control or obstructed bladders. These results suggest that the active electrical properties of the detrusor smooth muscle membrane are changed significantly by obstruction-induced bladder hypertrophy. Furthermore, the results suggest that adrenergic and cholinergic neurotransmitters do not contribute to these changes in AP kinetics following obstruction. The changes in AP properties with outflow obstruction-induced bladder hypertrophy were compared with those previously reported for the hypertrophic myocardium and were discussed in relation to the known impaired contractile properties of obstructed bladder smooth muscle.     

Natriuretic peptide responsive, cyclic guanosine monophosphate producing structures in the guinea pig bladder

PURPOSE: We examined the localization of natriuretic peptide responsive, cyclic guanosine monophosphate producing cells in the guinea pig bladder. MATERIALS AND METHODS: The bladder was removed from male guinea pigs sacrificed by cervical dislocation. The lateral wall of the bladder was cut into strips 2 mm thick. The tissue pieces were incubated in the presence of human atrial natriuretic peptide, rat brain natriuretic peptide and C-type natriuretic peptide or the nitric oxide donor DEANO (diethylamine NONOate or 1,1-diethyl-2-hydroxy-2-nitrosohydrazine) (Sigma). Cyclic guanosine monophosphate immunoreactivity was localized using an antibody against formaldehyde fixed cyclic guanosine monophosphate. RESULTS: Atrial natriuretic peptide and brain natriuretic peptide stimulated cyclic guanosine monophosphate synthesis in suburothelial interstitial cells, whereas C-type natriuretic peptide was not effective. In contrast, DEANO stimulated cyclic guanosine monophosphate synthesis in urothelial umbrella cells, suburothelial interstitial cells, muscle interstitial cells and neurons. The effect of atrial natriuretic peptide and brain natriuretic peptide was not inhibited by ODQ (1H-[1, 2, 4]oxadiazolo[4-3a]quinoxalin-1-one), an inhibitor of nitric oxide responsive soluble guanylyl cyclase. CONCLUSIONS: To our knowledge our findings show for the first time a localized effect of atrial natriuretic peptide and brain natriuretic peptide to the suburothelial cells of the guinea pig bladder. These cells express the soluble guanylyl cyclase and particulate guanylyl cyclase-A isoforms. The specific physiological role of these cells is not known but it was suggested that they may be involved in the generation or modulation of sensation. The results imply a role for natriuretic peptide-cyclic guanosine monophosphate signaling in the processing of sensory information in the bladder.