ICC样细胞上P2X受体介导的嘌呤能信号在膀胱功能调控中的作用研究
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摘要
背景及目的:
迄今为止,有关逼尿肌收缩的神经调控机制尚未完全阐明,导致许多排尿异常的治疗缺乏针对性,疗效欠佳。膀胱虽为一平滑肌器官,但神经对膀胱的功能控制却具有类似于骨骼肌的随意控制的特点,与此同时膀胱又保留了类似于胃肠及心脏的不依赖于神经的自主活动特征,因此膀胱被定义为介于自主和非自主神经支配之间的特殊的平滑肌器官。从解剖上看,骨骼肌神经支配和平滑肌的神经支配方式是不同的:每个骨骼肌细胞至少受一个运动神经末梢的支配,通过神经—肌肉接头完成兴奋传递。在这种神经支配方式下,兴奋性神经冲动可使所支配的骨骼肌产生快速、协调的同步收缩;而平滑肌细胞与神经末梢之间没有类似神经—肌肉接头的结构,神经末稍与平滑肌间也并非一对一的关系。传统理论认为,在这种神经支配方式下,兴奋性神经冲动到达时分泌囊泡释放神经递质,这些递质通过细胞间液扩散,作用于所支配的细胞,从而实现神经对所支配器官的功能调节。已有研究表明,正常膀胱的逼尿肌细胞之间主要以传导机械信号的中间连接为主,其传导速度较慢。这就留下了难以解释的问题:神经递质从释放到作用到逼尿肌细胞存在时间延迟,且因距离不等,延迟的时间不同,那么排尿时逼尿肌的随主观意识即时启动,协调收缩是如何完成的呢?
胃肠道等空腔脏器的有关研究给我们以启示。现已证实胃肠道、输尿管等具有“自主”收缩活动特性的器官均具有源于器官本身的自发兴奋调控体系。这一体系包含三个重要组成部分,即起搏细胞、兴奋传递通路和效应细胞。神经通过对这一体系的影响,实现对器官的功能调节。而Cajal间质细胞(interstitial cells of Cajal, ICC)在上述器官的自发性收缩中起着非常重要的作用,它不仅作为胃肠道基本电节律(basic electrical rhythm, BER)的起搏者,而且是壁内神经信息向平滑肌传送的中转站,并对有关信号起整合作用。ICC保持正常的结构、功能及分布,对胃肠动力的产生和功能调控至关重要,其分布密度明显减少和细胞网络完整性被破坏与一些胃肠动力紊乱性疾病如Hirschsprung’s病和假性肠道梗阻的发生密切相关。近年来国内外学者相继在人、豚鼠的输尿管、膀胱、前列腺等组织器官内发现了形态与胃肠道ICC非常相似的ICC样细胞(ICC-like cells)。研究表明,人的膀胱内ICC样细胞主要分布于黏膜下层、逼尿肌肌束边缘和肌细胞之间,并且与支配膀胱的神经关系密切。可是生理情况下,膀胱并不表现出自发性收缩,那么膀胱内的ICC样细胞在膀胱功能调节中究竟发挥什么作用呢?我们推测膀胱内ICC样细胞极有可能作为神经对逼尿肌调控的“中转站”,通过其特有的网络状结构,将来自神经的兴奋迅速传导至整个膀胱,启动逼尿肌协调一致地收缩,完成排尿活动。另一方面,由于ICC样细胞与神经的特殊关系,在膀胱充盈过程中,可能作为充盈信号的“感受器”,参与膀胱感觉信号的整合。
目前研究显示,支配膀胱的神经包括胆碱能、肾上腺素能以及非肾上腺素能非胆碱能神经。早在1972年,Burnstock等就发现支配膀胱的副交感神经兴奋后会同时释放乙酰胆碱和ATP,据此提出了嘌呤能神经学说,自从嘌呤能受体在平滑肌、神经组织中被发现以来,嘌呤能神经在膀胱功能调控中的作用日趋受到重视。研究表明,ATP不仅作为神经递质由支配膀胱的副交感神经释放,并且在膀胱充盈过程中,受牵拉的膀胱上皮细胞也会释放ATP参与膀胱感觉功能的形成。
本课题以新近研究发现的膀胱ICC样细胞作为主要关注对象,以ATP介导的嘌呤能神经信号为切入点,从一全新的层面解读膀胱功能调控机制,具有十分重要的理论价值,并可望为膀胱功能异常治疗提供新策略。
方法:
本课题以成年SD大鼠为研究对象。第一部分我们从形态学上探讨ICC样细胞作为逼尿肌神经调控的中转站的可能性。首先,我们通过扫描电镜和透射电镜对成年大鼠膀胱间质细胞的超微结构进行观察;然后以c-Kit为ICC样细胞的特异性标记物,通过免疫荧光染色对成年大鼠膀胱ICC样细胞的分布进行研究。最后采用c-Kit和PGP9.5免疫荧光双标法观察大鼠膀胱ICC样细胞与神经末梢的关系。
第二部分我们主要从功能学的角度探讨ICC样细胞作为逼尿肌神经调控的“中转站”的可能性。我们分别从ATP对正常大鼠离体逼尿肌条收缩功能的影响、ATP对原代培养的大鼠膀胱ICC样细胞及DSMC内Ca2+的影响、大鼠膀胱ICC样细胞及DSMC上P2X受体亚型分布等不同角度进行分析研究。
第三部分我们通过膀胱内灌注Glevic阻断ICC样细胞功能对大鼠盆神经传入放电的影响,以探讨ICC样细胞在膀胱感觉传入中的作用。
结果:
1.扫描电镜观察显示,去除黏膜层以后,膀胱黏膜下层可见部分间质细胞,胞体小,呈圆形或多角形,伸出数个细长突起,相互连接呈网络状。
2.透射电镜下观察显示,大鼠膀胱逼尿肌肌层间质细胞胞膜不完整,存在胞膜小凹,胞质和突起内含丰富的线粒体,胞浆中有较多中间丝。间质细胞为逼尿肌细胞所环绕,二者胞膜之间紧密相邻处可见多个斑状结构,形成典型缝隙连接;在局部区域区可见间质细胞的突起与神经末梢联系紧密。
3.激光共聚焦显微镜下观察显示:SD大鼠膀胱黏膜下层、肌束边缘以及肌束间均可见c-Kit染色阳性的ICC样细胞;黏膜下层的ICC样细胞相互连接呈网络状。
4.免疫荧光双标显示, SD大鼠膀胱内PGP9.5阳性的神经末梢和c-Kit阳性的ICC样细胞的突起接触紧密。
5.在一定的前负荷(0.3g)下,离体的逼尿肌条可出现期相性收缩。浓度高于10μmol/L的ATP可诱发肌条出现先收缩后舒张的双相效应;收缩效应及舒张效应均随ATP浓度增加而增强,1 mmol/L ATP作用后肌条在一过性收缩后持续处于舒张状态。
6.成功进行了大鼠膀胱ICC样细胞和DSMC的原代混合培养及鉴定。
7. Fluo-4/NW染色后,激光共聚焦显微镜观察显示,静止状态下ICC样细胞内[Ca2+]i平均荧光强度(55.16±3.23)显著强于平滑肌细胞(45.59±2.08)(P<0.05);20μM和100μM ATP可诱发体外培养的ICC样细胞及DSMC细胞内[Ca2+]i一过性升高,随后缓慢下降。
8.免疫组化染色显示SD大鼠膀胱组织可表达7种P2X受体亚型,逼尿肌细胞可见P2X1亚型表达,免疫荧光双标显示ICC样细胞可表达P2X2和P2X5。
9.膀胱内灌注ATP(终浓度20mM)可诱发大鼠盆神经放电,频率为28.47±3.97次/分;而预先灌注100μM glevic阻断ICC样细胞功能后,ATP诱发的盆神经放电频率下降至2.35±0.42次/分,明显低于ATP组(P<0.01)。
结论:
1.形态学研究提示大鼠膀胱ICC样细胞具备作为神经末梢调控逼尿肌细胞的“中转站”的结构基础。
2. ATP对离体的逼尿肌条出现先收缩后舒张的双相效应,表明嘌呤能信号在膀胱功能调控中起重要作用。
3. ATP可诱发ICC样细胞和DSMC内[Ca2+]i明显升高,DSMC上的P2X1受体和ICC样细胞上的P2X2和P2X5受体是嘌呤能神经递质ATP激活DSMC和ICC样细胞的重要分子基础。
4.膀胱内灌注ATP可诱发大鼠盆神经放电,阻断ICC样细胞功能后,盆神经放电频率明显降低,提示ICC样细胞极有可能参与了ATP介导的膀胱感觉传入。
Background and Aim: Up to now, the therapies of many voiding disorders are unspecific and the curative effects are unsatisfactory because the mechanisms of urinary bladder neuromodulation are not elucidated completely. As a smooth muscle organ, bladder not only kept the independence character of smooth muscle organs, such as gastrointestinal tract, but also gained the adlibitum control character of somatic muscles. How this two physiological character cooperate each other are not clear. So bladder was defined as a special smooth muscle organ. The traditional neuromodulation mode of smooth muscle can not be used to explain the complicate detrusor neuroregulation exactly.
In the gastrointestinal tract, the interstitial cells of Cajal (ICC) have important physiological functions, including pacemaking, relaying of nerve impulses and propagation of excitation. In the last few years,Several papers have described the presence of ICC-like cells throughout the urinary tract. Bladder ICC-like cells are recognized to lie in two predominant anatomical locations, beneath the urothelium and within the detrusor muscle, in both the human and guinea-pig. While under physiological condition, spontaneous contraction was not observed during urine storage. So it is very intriguing to investigate the functional properties of bladder ICC-like cells. In this study, we speculate that ICC-like cells in bladder are contributed to the propagation of excitation from the nerve terminals to detrusor smooth muscle,just be a‘relay station’more than a‘pacemaker’. As we know, the motility of bladder is under control of the nervous system.
Sympathetic, parasympathetic, somatic and sensory pathways contribute to the control of bladder during urine storage and micturition. In addition to the classical neurotransmitters noradrenaline and acetylcholine, peptides, purines and the enzyme responsible for the synthesis of nitric oxide have all been localized in nerves supplying the bladder. As early as 1972 Burnstock et al postulated that ATP can act as the excitatory co-transmitter with acetylcholine in parasympathetic nerves supplying the urinary bladder. It was later shown that, indeed, ATP is released with acetylcholine (Ach) in response to nerve stimulation and that the ATP response is mediated through ligand-gated cation channels, P2X receptors.
In this study, we focused on the role of ICC-like cells in bladder neuromodulation, incisivused with purinergic signal mediated by P2X receptor. The results are helpful to enrich neurophysiology theory of the bladder, important for understanding of the physiology and pathology of micturition and to provide new targets for the therapy of some voiding dysfunctions.
Methods: 64 SD rats and 1 guinea pig were sacrificed in this study. Firstly, we investigated the ultrastructure of interstitial cells beneath the urothelium and within the detrusor muscle of SD rat by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).On the second, immunoflurorescence were used to identify ICC-like cells with c-Kit as the markers in SD rat bladder and then immunoflurorescence (double-label) was used to study the relationship between ICC-like cells and nerve terminals. Thirdly, ICC-like cells and detrusor smooth muscle cells (DSMC) were isolated by collagenase digestion and co-cultured in vitro, then the effect of ATP on [Ca2+]i variation of ICC-like cells and DSMC were assessed by confocal laser scanning microscopy (CLSM),then immunohistochemistry and immunoflurorescence (double-label)were used to study the expression of P2X purinoceptor subtypes in DSMC and ICC-like cells respectively. In the third part, to evaluate the role ICC-like cells in urinary bladder sensation, the variation of rat pelvic nerve afferent discharge evoked by filling ATP into the urinary bladder before and after blocking the function of ICC-like cells with Glevic。
Main results:
1. SEM showed us some interstitial cells beneath the urothelium with the similar morphological features as follow, small round or polygon cell body and several slender ecphyma stretching out. They connected with each other and constructed a cell-net.
2. With TEM, we found the morphological features of interstitial cells within the detrusor muscles, such as gap junctions with DSMCs and very close apposition with nerve terminals.
3. ICC-like cells were identified to exist in suburothlium layer,along the longititude of smooth muscle bundles and among detrusor smooth muscle in adult SD rat urinary bladder through c-Kit immunofluorescence.
4. Double-labelled tissue with c-Kit and PGP9.5 antibodies showed that ICC-like cells are very close apposition with nerve terminals in SD rat bladder.
5. Spontaneous contractions were recorded on detrusor smooth muscle strips in vitro under certain preload (0.5g. When ATP at concentrations greater than 10μM was added, a transient contraction followed by long time diastole was recorded on detrusor smooth muscle strips in vitro, the dose-effect relationship of ATP is obviously. The spontaneous contractile amplitude was vanished completely by 1 mmol/L ATP.
6. We established the primary culture method of ICC-like cells and DSMCs in vitro by collagenase digestion.
7. The concentration of intracellular Ca2+ ion of ICC-like cells and DSMCs were detected in dynamic state by LSCM, the mean intensity of [Ca2+]i of ICC-like cells (55.16±3.23)is higher than that of DSMCs(45.59±2.08)(P<0.05). Continuous application of ATP at concentrations greater than 20μM caused a prompt rise followed by a slow decline of the [Ca2+]i baseline in both ICC-like cells and DSMCs.
8. Immunohistochemistry results showed us that all seven subtype of P2X receptor expressed in SD rat urinary bladder, among them, only P2X1 was distributed on DSMCs. P2X2 and P2X5 were detected on ICC-like cells by immunoflurorescence (double-label).
9. Filling with 20 mM ATP into the urinary bladder can evoke SD rat pelvic nerve afferent activity. When blocking the function of ICC-like cells by 100μM Glevic in advance , the pelvic nerve afferent activity evoked by ATP was obviously decreased(P<0.01)。
Conclusions:
1. Morphological-study hinted that ICC-like cells in rat urinary bladder owned the ability of“relay station”between the nerve terminals and DSMCs.
2. The effects of ATP on detrusor smooth muscle strips indicated that purinergic signal is very important for detrusor neuromodulation.
3. Our results of ATP on intracellular Ca2+ ion of ICC-like cells and DSMCs and the difference expression of P2X receptor on ICC-like cells and DSMCs showed us that ATP can excite ICC-like cells and DSMCs through different P2X receptor subtypes.
4. The effects of Glevic on rat pelvic nerve afferent discharge evoked by ATP indicated that ICC-like cells may play very important role in bladder sensation.
迄今为止,有关逼尿肌收缩的神经调控机制尚未完全阐明,导致许多排尿异常的治疗缺乏针对性,疗效欠佳。膀胱虽为一平滑肌器官,但神经对膀胱的功能控制却具有类似于骨骼肌的随意控制的特点,与此同时膀胱又保留了类似于胃肠及心脏的不依赖于神经的自主活动特征,因此膀胱被定义为介于自主和非自主神经支配之间的特殊的平滑肌器官。从解剖上看,骨骼肌神经支配和平滑肌的神经支配方式是不同的:每个骨骼肌细胞至少受一个运动神经末梢的支配,通过神经—肌肉接头完成兴奋传递。在这种神经支配方式下,兴奋性神经冲动可使所支配的骨骼肌产生快速、协调的同步收缩;而平滑肌细胞与神经末梢之间没有类似神经—肌肉接头的结构,神经末稍与平滑肌间也并非一对一的关系。传统理论认为,在这种神经支配方式下,兴奋性神经冲动到达时分泌囊泡释放神经递质,这些递质通过细胞间液扩散,作用于所支配的细胞,从而实现神经对所支配器官的功能调节。已有研究表明,正常膀胱的逼尿肌细胞之间主要以传导机械信号的中间连接为主,其传导速度较慢。这就留下了难以解释的问题:神经递质从释放到作用到逼尿肌细胞存在时间延迟,且因距离不等,延迟的时间不同,那么排尿时逼尿肌的随主观意识即时启动,协调收缩是如何完成的呢?
胃肠道等空腔脏器的有关研究给我们以启示。现已证实胃肠道、输尿管等具有“自主”收缩活动特性的器官均具有源于器官本身的自发兴奋调控体系。这一体系包含三个重要组成部分,即起搏细胞、兴奋传递通路和效应细胞。神经通过对这一体系的影响,实现对器官的功能调节。而Cajal间质细胞(interstitial cells of Cajal, ICC)在上述器官的自发性收缩中起着非常重要的作用,它不仅作为胃肠道基本电节律(basic electrical rhythm, BER)的起搏者,而且是壁内神经信息向平滑肌传送的中转站,并对有关信号起整合作用。ICC保持正常的结构、功能及分布,对胃肠动力的产生和功能调控至关重要,其分布密度明显减少和细胞网络完整性被破坏与一些胃肠动力紊乱性疾病如Hirschsprung’s病和假性肠道梗阻的发生密切相关。近年来国内外学者相继在人、豚鼠的输尿管、膀胱、前列腺等组织器官内发现了形态与胃肠道ICC非常相似的ICC样细胞(ICC-like cells)。研究表明,人的膀胱内ICC样细胞主要分布于黏膜下层、逼尿肌肌束边缘和肌细胞之间,并且与支配膀胱的神经关系密切。可是生理情况下,膀胱并不表现出自发性收缩,那么膀胱内的ICC样细胞在膀胱功能调节中究竟发挥什么作用呢?我们推测膀胱内ICC样细胞极有可能作为神经对逼尿肌调控的“中转站”,通过其特有的网络状结构,将来自神经的兴奋迅速传导至整个膀胱,启动逼尿肌协调一致地收缩,完成排尿活动。另一方面,由于ICC样细胞与神经的特殊关系,在膀胱充盈过程中,可能作为充盈信号的“感受器”,参与膀胱感觉信号的整合。
目前研究显示,支配膀胱的神经包括胆碱能、肾上腺素能以及非肾上腺素能非胆碱能神经。早在1972年,Burnstock等就发现支配膀胱的副交感神经兴奋后会同时释放乙酰胆碱和ATP,据此提出了嘌呤能神经学说,自从嘌呤能受体在平滑肌、神经组织中被发现以来,嘌呤能神经在膀胱功能调控中的作用日趋受到重视。研究表明,ATP不仅作为神经递质由支配膀胱的副交感神经释放,并且在膀胱充盈过程中,受牵拉的膀胱上皮细胞也会释放ATP参与膀胱感觉功能的形成。
本课题以新近研究发现的膀胱ICC样细胞作为主要关注对象,以ATP介导的嘌呤能神经信号为切入点,从一全新的层面解读膀胱功能调控机制,具有十分重要的理论价值,并可望为膀胱功能异常治疗提供新策略。
方法:
本课题以成年SD大鼠为研究对象。第一部分我们从形态学上探讨ICC样细胞作为逼尿肌神经调控的中转站的可能性。首先,我们通过扫描电镜和透射电镜对成年大鼠膀胱间质细胞的超微结构进行观察;然后以c-Kit为ICC样细胞的特异性标记物,通过免疫荧光染色对成年大鼠膀胱ICC样细胞的分布进行研究。最后采用c-Kit和PGP9.5免疫荧光双标法观察大鼠膀胱ICC样细胞与神经末梢的关系。
第二部分我们主要从功能学的角度探讨ICC样细胞作为逼尿肌神经调控的“中转站”的可能性。我们分别从ATP对正常大鼠离体逼尿肌条收缩功能的影响、ATP对原代培养的大鼠膀胱ICC样细胞及DSMC内Ca2+的影响、大鼠膀胱ICC样细胞及DSMC上P2X受体亚型分布等不同角度进行分析研究。
第三部分我们通过膀胱内灌注Glevic阻断ICC样细胞功能对大鼠盆神经传入放电的影响,以探讨ICC样细胞在膀胱感觉传入中的作用。
结果:
1.扫描电镜观察显示,去除黏膜层以后,膀胱黏膜下层可见部分间质细胞,胞体小,呈圆形或多角形,伸出数个细长突起,相互连接呈网络状。
2.透射电镜下观察显示,大鼠膀胱逼尿肌肌层间质细胞胞膜不完整,存在胞膜小凹,胞质和突起内含丰富的线粒体,胞浆中有较多中间丝。间质细胞为逼尿肌细胞所环绕,二者胞膜之间紧密相邻处可见多个斑状结构,形成典型缝隙连接;在局部区域区可见间质细胞的突起与神经末梢联系紧密。
3.激光共聚焦显微镜下观察显示:SD大鼠膀胱黏膜下层、肌束边缘以及肌束间均可见c-Kit染色阳性的ICC样细胞;黏膜下层的ICC样细胞相互连接呈网络状。
4.免疫荧光双标显示, SD大鼠膀胱内PGP9.5阳性的神经末梢和c-Kit阳性的ICC样细胞的突起接触紧密。
5.在一定的前负荷(0.3g)下,离体的逼尿肌条可出现期相性收缩。浓度高于10μmol/L的ATP可诱发肌条出现先收缩后舒张的双相效应;收缩效应及舒张效应均随ATP浓度增加而增强,1 mmol/L ATP作用后肌条在一过性收缩后持续处于舒张状态。
6.成功进行了大鼠膀胱ICC样细胞和DSMC的原代混合培养及鉴定。
7. Fluo-4/NW染色后,激光共聚焦显微镜观察显示,静止状态下ICC样细胞内[Ca2+]i平均荧光强度(55.16±3.23)显著强于平滑肌细胞(45.59±2.08)(P<0.05);20μM和100μM ATP可诱发体外培养的ICC样细胞及DSMC细胞内[Ca2+]i一过性升高,随后缓慢下降。
8.免疫组化染色显示SD大鼠膀胱组织可表达7种P2X受体亚型,逼尿肌细胞可见P2X1亚型表达,免疫荧光双标显示ICC样细胞可表达P2X2和P2X5。
9.膀胱内灌注ATP(终浓度20mM)可诱发大鼠盆神经放电,频率为28.47±3.97次/分;而预先灌注100μM glevic阻断ICC样细胞功能后,ATP诱发的盆神经放电频率下降至2.35±0.42次/分,明显低于ATP组(P<0.01)。
结论:
1.形态学研究提示大鼠膀胱ICC样细胞具备作为神经末梢调控逼尿肌细胞的“中转站”的结构基础。
2. ATP对离体的逼尿肌条出现先收缩后舒张的双相效应,表明嘌呤能信号在膀胱功能调控中起重要作用。
3. ATP可诱发ICC样细胞和DSMC内[Ca2+]i明显升高,DSMC上的P2X1受体和ICC样细胞上的P2X2和P2X5受体是嘌呤能神经递质ATP激活DSMC和ICC样细胞的重要分子基础。
4.膀胱内灌注ATP可诱发大鼠盆神经放电,阻断ICC样细胞功能后,盆神经放电频率明显降低,提示ICC样细胞极有可能参与了ATP介导的膀胱感觉传入。
Background and Aim: Up to now, the therapies of many voiding disorders are unspecific and the curative effects are unsatisfactory because the mechanisms of urinary bladder neuromodulation are not elucidated completely. As a smooth muscle organ, bladder not only kept the independence character of smooth muscle organs, such as gastrointestinal tract, but also gained the adlibitum control character of somatic muscles. How this two physiological character cooperate each other are not clear. So bladder was defined as a special smooth muscle organ. The traditional neuromodulation mode of smooth muscle can not be used to explain the complicate detrusor neuroregulation exactly.
In the gastrointestinal tract, the interstitial cells of Cajal (ICC) have important physiological functions, including pacemaking, relaying of nerve impulses and propagation of excitation. In the last few years,Several papers have described the presence of ICC-like cells throughout the urinary tract. Bladder ICC-like cells are recognized to lie in two predominant anatomical locations, beneath the urothelium and within the detrusor muscle, in both the human and guinea-pig. While under physiological condition, spontaneous contraction was not observed during urine storage. So it is very intriguing to investigate the functional properties of bladder ICC-like cells. In this study, we speculate that ICC-like cells in bladder are contributed to the propagation of excitation from the nerve terminals to detrusor smooth muscle,just be a‘relay station’more than a‘pacemaker’. As we know, the motility of bladder is under control of the nervous system.
Sympathetic, parasympathetic, somatic and sensory pathways contribute to the control of bladder during urine storage and micturition. In addition to the classical neurotransmitters noradrenaline and acetylcholine, peptides, purines and the enzyme responsible for the synthesis of nitric oxide have all been localized in nerves supplying the bladder. As early as 1972 Burnstock et al postulated that ATP can act as the excitatory co-transmitter with acetylcholine in parasympathetic nerves supplying the urinary bladder. It was later shown that, indeed, ATP is released with acetylcholine (Ach) in response to nerve stimulation and that the ATP response is mediated through ligand-gated cation channels, P2X receptors.
In this study, we focused on the role of ICC-like cells in bladder neuromodulation, incisivused with purinergic signal mediated by P2X receptor. The results are helpful to enrich neurophysiology theory of the bladder, important for understanding of the physiology and pathology of micturition and to provide new targets for the therapy of some voiding dysfunctions.
Methods: 64 SD rats and 1 guinea pig were sacrificed in this study. Firstly, we investigated the ultrastructure of interstitial cells beneath the urothelium and within the detrusor muscle of SD rat by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).On the second, immunoflurorescence were used to identify ICC-like cells with c-Kit as the markers in SD rat bladder and then immunoflurorescence (double-label) was used to study the relationship between ICC-like cells and nerve terminals. Thirdly, ICC-like cells and detrusor smooth muscle cells (DSMC) were isolated by collagenase digestion and co-cultured in vitro, then the effect of ATP on [Ca2+]i variation of ICC-like cells and DSMC were assessed by confocal laser scanning microscopy (CLSM),then immunohistochemistry and immunoflurorescence (double-label)were used to study the expression of P2X purinoceptor subtypes in DSMC and ICC-like cells respectively. In the third part, to evaluate the role ICC-like cells in urinary bladder sensation, the variation of rat pelvic nerve afferent discharge evoked by filling ATP into the urinary bladder before and after blocking the function of ICC-like cells with Glevic。
Main results:
1. SEM showed us some interstitial cells beneath the urothelium with the similar morphological features as follow, small round or polygon cell body and several slender ecphyma stretching out. They connected with each other and constructed a cell-net.
2. With TEM, we found the morphological features of interstitial cells within the detrusor muscles, such as gap junctions with DSMCs and very close apposition with nerve terminals.
3. ICC-like cells were identified to exist in suburothlium layer,along the longititude of smooth muscle bundles and among detrusor smooth muscle in adult SD rat urinary bladder through c-Kit immunofluorescence.
4. Double-labelled tissue with c-Kit and PGP9.5 antibodies showed that ICC-like cells are very close apposition with nerve terminals in SD rat bladder.
5. Spontaneous contractions were recorded on detrusor smooth muscle strips in vitro under certain preload (0.5g. When ATP at concentrations greater than 10μM was added, a transient contraction followed by long time diastole was recorded on detrusor smooth muscle strips in vitro, the dose-effect relationship of ATP is obviously. The spontaneous contractile amplitude was vanished completely by 1 mmol/L ATP.
6. We established the primary culture method of ICC-like cells and DSMCs in vitro by collagenase digestion.
7. The concentration of intracellular Ca2+ ion of ICC-like cells and DSMCs were detected in dynamic state by LSCM, the mean intensity of [Ca2+]i of ICC-like cells (55.16±3.23)is higher than that of DSMCs(45.59±2.08)(P<0.05). Continuous application of ATP at concentrations greater than 20μM caused a prompt rise followed by a slow decline of the [Ca2+]i baseline in both ICC-like cells and DSMCs.
8. Immunohistochemistry results showed us that all seven subtype of P2X receptor expressed in SD rat urinary bladder, among them, only P2X1 was distributed on DSMCs. P2X2 and P2X5 were detected on ICC-like cells by immunoflurorescence (double-label).
9. Filling with 20 mM ATP into the urinary bladder can evoke SD rat pelvic nerve afferent activity. When blocking the function of ICC-like cells by 100μM Glevic in advance , the pelvic nerve afferent activity evoked by ATP was obviously decreased(P<0.01)。
Conclusions:
1. Morphological-study hinted that ICC-like cells in rat urinary bladder owned the ability of“relay station”between the nerve terminals and DSMCs.
2. The effects of ATP on detrusor smooth muscle strips indicated that purinergic signal is very important for detrusor neuromodulation.
3. Our results of ATP on intracellular Ca2+ ion of ICC-like cells and DSMCs and the difference expression of P2X receptor on ICC-like cells and DSMCs showed us that ATP can excite ICC-like cells and DSMCs through different P2X receptor subtypes.
4. The effects of Glevic on rat pelvic nerve afferent discharge evoked by ATP indicated that ICC-like cells may play very important role in bladder sensation.
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