【作者】 郑霁；

【导师】 宋波；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 背景及目的:膀胱逼尿肌是一种特殊的平滑肌组织,逼尿肌组织在受到神经支配的同时,是否存在类似心脏和胃肠道平滑肌的自发性兴奋,曾经是个认识不清的问题。我科对之进行了较系统的研究后发现:①离体条件下膀胱逼尿肌肌条可出现自发的兴奋性收缩;②离体膀胱逼尿肌条在多种神经递质拮抗剂“鸡尾酒”式的混合阻断下,仍然能产生收缩。以上研究结果及国外相关研究让我们认识到:逼尿肌组织具有自主产生兴奋的能力,这种自发性肌源性兴奋并不依赖于神经因素的作用而存在。我们的实验研究还发现,在膀胱流出道梗阻和神经源性异常引起的逼尿肌不稳定（Detrusor instability,DI）时,这种肌源性的自发兴奋和收缩明显增强,表现为:不稳定逼尿肌肌条与正常逼尿肌肌条相比,自发性收缩的幅度和频率明显增加;同等张力负荷下不稳定逼尿肌肌条的自发性收缩频率较正常逼尿肌肌条明显增加。膀胱逼尿肌兴奋性增高亦常见于临床,如继发于神经源性膀胱功能障碍及膀胱出口梗阻时。这一异常现象的发生目前被归咎为两个基本的机制:①神经源性学说。这一学说居主导地位,以de Groat为代表,强调所有兴奋性改变均源于神经因素,或为神经因素的延续效应,主要是表现在神经受体的数量和功能的上调。②肌源性学说。以Brading为代表,认为兴奋性改变除神经因素作用外,尚有肌源性异常因素参与,包括逼尿肌肌细胞的兴奋性增高、细胞间信号传导功能的加强、逼尿肌细胞与神经节细胞的联系增加,以及膀胱内可能存在的起搏细胞功能数量及功能的上调等。对膀胱肌源性兴奋的机制进行研究将有望为相关膀胱功能障碍性疾病开创新的治疗策略提供理论基础。在膀胱兴奋的肌源性学说这一理论中,膀胱内是否存在具有起搏功能的细胞至关重要,也一直为人们所重视。Cajal间质细胞（Interstitial cells of cajal,ICC）是一类与平滑肌细胞在形态及功能上均不相同细胞,业已证明这些细胞是胃肠道、输尿管等器官的起搏细胞,这些器官的平滑肌自发性兴奋均起源于其中ICCs细胞。McCloskey等及Shafik等采用c-kit特异性标记的方法,先后证实了在豚鼠和人的膀胱中存在ICCs细胞,这种细胞呈长梭形,在去极化时不能产生自发收缩,有别于正常的逼尿肌肌细胞,更为特异的是这种细胞c-kit表达呈阳性,它们主要沿肌束分布,并可产生钙活动,与神经和平滑肌细胞也有广泛的联系,极可能是起搏细胞或是兴奋调节细胞。后续ICCs细胞功能方面的研究发现,ICCs细胞具有自动除极的外向电流,c-kit的特异性阻断剂Glivec可显著抑制逼尿平滑肌束的自发性动作电位和兴奋收缩能力;提示ICCs细胞可能具备自动除极化,完成自动起搏的能力,这从功能学上肯定了ICCs细胞的存在以及其作为起搏细胞的可能性。但ICCs细胞是否是膀胱逼尿肌自发性兴奋的起搏细胞,目前尚无明确结论;开展膀胱逼尿肌组织中相关研究,特别对ICCs细胞和平滑肌细胞的自发性兴奋特征进行对比,将为阐明膀胱ICCs细胞是否是膀胱起搏细胞及其可能具有的功能提供重要的研究证据。钙离子是细胞内信号转导重要的第二信使,在平滑肌细胞中,钙自发性释放入胞浆导致的钙火花(Ca²⁺ spark)及细胞外钙内流诱导内质网中钙释放(Ca²⁺ induced Ca²⁺ release, CICR)均是通过Ⅱ型Ryanodine受体（RyR2）完成的。细胞钙瞬变(Ca²⁺ transients)与动作电位的发生关系密切,是动作电位发生的重要信号。动作电位相关的平滑肌细胞膜去极化可致细胞膜上电压门控的钙离子通道开放,细胞外钙内流随后通过CICR作用诱导细胞内质网（sarcoplasmic reticulum,SR）中的钙释放入胞浆,导致细胞浆内钙离子浓度瞬间升高,引起细胞发生钙瞬变和平滑肌收缩。环腺苷二磷酸核糖（Cyclic adenosine diphosphate ribose,cADPR）是细胞内烟酰胺腺嘌呤二核苷酸（NAD+）的代谢产物,是目前为止除钙离子外,平滑肌细胞内对细胞内质网上RyR2有调节作用的又一重要第二信使。以往的研究发现,cADPR可通过调控平滑肌细胞内质网RyR2开放对平滑肌细胞钙瞬变的发生及钙瞬变特征产生显著调节作用,其具体机制为:cADPR可通过影响细胞自发性瞬时外向电流（spontaneous transient outward currents,STOCs）、自发性瞬时内向电流（spontaneous transient outward currents,STICs）影响平滑肌细胞去极化,并可通过影响钙诱导的钙释放（CICR）影响平滑肌细胞细胞钙瞬变的幅度。但目前cADPR调节平滑肌细胞内质网钙释放的作用机制仍不清楚,研究结论还存在较大的矛盾。探讨cADPR调节平滑肌细胞内质网中钙释放这一cADPR调控细胞钙瞬变发生关键环节的机制,将为深入了解cADPR调控平滑肌细胞钙瞬变的过程提供实验依据。考虑到细胞钙瞬变与细胞动作电位的发生密切相关,cADPR可通过影响细胞内质网中钙释放进而调控平滑肌钙瞬变的发生及其特征,本研究拟以细胞钙瞬变为观察指标,比较分析ICCs细胞和平滑肌细胞自发性钙瞬变的特征及发生机制;并以反映平滑肌细胞内钙释放的STOCs和钙火花(Ca²⁺ spark)为观察指标,通过利用FKBP12.6基因敲除小鼠,对cADPR调节平滑肌细胞内质网中钙释放的机制进行探讨。本研究将有助于我们深入对平滑肌细胞和ICCs细胞自发性钙瞬变特征、发生和调控的认识,并为阐明膀胱ICCs细胞的功能提供初步的研究证据。本研究拟从以下四个方面展开:第一:利用免疫荧光单染及双染的方法,观察ICCs细胞在逼尿肌组织中的分布及与平滑肌细胞的关系,为后续平滑肌细胞和ICCs细胞自发性钙瞬变的检测提供参考;第二:对膀胱逼尿肌组织铺片中的平滑肌细胞和相邻ICCs细胞自发性钙瞬变进行检测,比较分析两种细胞自发性钙瞬变的频率和幅度,初步探讨平滑肌相邻ICCs细胞可能的功能;第三:检测细胞外钙内流及细胞钙库中钙释放干预因素对细胞自发性钙瞬变的影响,探讨细胞外钙内流及细胞内质网中钙释放入胞浆在两种细胞自发性钙瞬变发生的作用;第四:对比野生型和FKBP12.6基因敲除小鼠中cADPR对膀胱平滑肌细胞STOCs、钙火花及细胞内质网总钙含量的影响,探讨cADPR调节平滑肌细胞内质网中钙释放这一cADPR调控细胞钙瞬变发生关键环节的机制,为深入了解cADPR调控平滑肌细胞钙瞬变的机制提供实验依据。方法:本研究采用2-3月龄SD大鼠为研究对象。首先利用免疫荧光方法检测逼尿肌组织中ICCs细胞的分布及其与平滑肌细胞的关系。随后参照Hashitani等[18]的方法制作膀胱组织铺片,于记录Chamber底部Physiological saline生理液灌流保持肌条铺片活性。待肌条自发性收缩活动出现后,Fluo-4 AM负载肌条铺片,激光共聚焦显微镜观察铺片中平滑肌细胞和ICCs细胞自发性钙瞬变,随后分别观察L型、T型钙离子通道阻断剂Nimodipine、Nicl2,内质网IP3受体拮抗剂2-aminoethoxy diphenylborate （2-APB）,内质网RyR受体拮抗剂Ryanodine,内质网RyR受体激动剂Caffeine,内质网钙泵抑制剂Thapsigargin及无钙、高钙生理液对平滑肌细胞和ICCs细胞自发性钙瞬变的影响,探讨平滑肌细胞和ICCs细胞自发性钙瞬变产生、调控的机制。最后利用FKBP12.6基因敲除小鼠探讨cADPR调节膀胱平滑肌细胞内质网中钙释放的机制。通过PCR方法对敲除小鼠的基因表型进行鉴定。通过膜片钳记录cADPR对平滑肌细胞自发性瞬时外向电流（spontaneous transient out currents,STOCs）的影响,使用膜片钳和激光共聚焦显微镜共记录的方法,同步观察cADPR对平滑肌细胞STOCs和钙火花(Ca²⁺ spark)的影响。利用激光共聚焦显微镜观察cADPR对膀胱平滑肌细胞细胞钙火花特征的影响,并通过Thapsigargin预处理了解内质网钙泵是否参与cADPR调节平滑肌细胞中钙释放作用。利用Western blot实验,检测cADPR作用后,FKBP12.6从RyR2上释放入胞浆的情况。利用双Puff系统,通过瞬时使用10mM Caffeine,探讨cADPR对平滑肌细胞内质网中总钙含量的影响。结果:1. SD大鼠膀胱逼尿肌组织中存在ICCs细胞,ICCs细胞在逼尿肌肌束旁、肌束间和肌束内均有分布;ICCs细胞与平滑肌细胞、神经末梢在空间结构上紧密相邻;肌束旁和肌束间的ICCs细胞有时可连成一片,有可能成为相邻肌束之间联系的“桥梁”;2.逼尿肌组织铺片中平滑肌细胞自发性钙瞬变的特征如下:（1）逼尿肌组织铺片Fluo-4刚负载完成时,平滑肌束中平滑肌细胞自发性钙瞬变多不同步;热的生理液灌流20-30min后,平滑肌束中平滑肌细胞自发性钙瞬变趋于同步;（2）平滑肌细胞钙瞬变多起源于肌束边缘,而后传递到肌束的另一侧;部分钙瞬变可起源于肌束中部,而后向肌束两侧传递;（3）平滑肌细胞钙瞬变在肌束内纵向传递几乎无时间延迟,但其在肌束内横向传递有一定的时间延迟;（4）观察到相邻的肌束间可通过肌束间细胞进行钙瞬变的传递。3.成功同时记录到膀胱逼尿肌组织铺片中平滑肌细胞和肌束旁ICCs细胞的自发性钙瞬变,平滑肌细胞自发性钙瞬变频率相对较快,而肌束旁ICCs细胞的自发性钙瞬变频率相对较慢;4.膀胱逼尿肌组织铺片中的药理实验研究发现:（1） L型钙离子通道阻断剂Nimodipine能完全抑制平滑肌细胞的自发性钙瞬变,但对ICCs细胞的自发性钙瞬变无显著影响;T型钙离子通道阻断剂Nicl2可明显降低平滑肌细胞的自发性钙瞬变的频率,对平滑肌细胞钙瞬变的幅度及ICCs细胞的自发性钙瞬变无明显影响;（2） Ryanodine,Caffeine,2-APB,和Thapsigargin均可对平滑肌细胞的自发性钙瞬变产生显著的抑制作用,但不能完全消除平滑肌细胞的自发性钙瞬变的发生;（3） Ryanodine,Caffeine,2-APB,和Thapsigargin及无钙生理液均可完全抑制ICCs细胞的自发性钙瞬变,高钙生理液可显著增加ICCs细胞自发性钙瞬变的频率而降低其频率。5.成功对FKBP12.6基因敲除小鼠进行了基因表型鉴定。经鉴定,基因敲除小鼠繁殖后,表现为三种基因表型:纯合子（FKBP12.6 -/-）、杂合子（FKBP12.6 +/-）和野生型（FKBP12.6 +/+）;6. cADPR可显著增大野生型小鼠膀胱平滑肌细胞的STOCs,对FKBP12.6基因敲除小鼠（纯和子）平滑肌细胞STOCs却无明显影响;7.膜片钳和激光共聚焦显微镜共记录的研究结果发现,cADPR可显著增强野生型小鼠膀胱平滑肌细胞的STOCs及钙火花,但对FKBP12.6基因敲除小鼠平滑肌细胞STOCs及钙火花却无明显影响;8. cADPR对野生型小鼠膀胱平滑肌细胞钙火花有显著影响,而对FKBP12.6基因敲除小鼠平滑肌细胞钙火花却无明显调节作用。具体表现为:cADPR可显著提高野生型小鼠膀胱平滑肌细胞钙火花的频率、幅度及FWHM距离,明显延长其上升时间和半数衰减时间;而cADPR对FKBP12.6基因敲除小鼠细胞钙火花的上述特征性指标无明显影响;9.内质网钙泵抑制剂Thapsigargin对cADPR调节平滑肌细胞自发性钙释放的作用无明显影响;10. Western blot实验证实,cADPR可使FKBP12.6从RyR2上脱落下来,释放入胞浆中;11. cADPR可显著降低野生型小鼠膀胱平滑肌细胞内质网中的总钙含量,而对FKBP12.6基因敲除小鼠内质网中的总钙含量却无明显影响。结论:1.膀胱平滑肌自发性钙瞬变在肌束中纵向传递和横向传递的延迟不同可能来自传递机制的不同,而肌束间钙瞬变的传递增强可能参与DI的发生;2.热生理液灌流使肌束中平滑肌细胞自发性钙瞬变趋于同步及钙瞬变多起源于肌束边缘提示:膀胱逼尿肌组织中存在起搏细胞的可能性很大,可能是膀胱起搏细胞的是:①肌束一侧的平滑肌细胞和②肌束旁或肌束内ICCs细胞;钙瞬变也可起源于肌束中部,提示不同部位的膀胱平滑肌组织起搏细胞的分布可能存在差异;3.膀胱组织铺片中平滑肌细胞自发性钙瞬变频率明显快于肌束旁ICCs细胞,提示平滑肌细胞自发性钙瞬变起源于肌束旁ICCs细胞的可能性不大;考虑到目前肌束内ICCs细胞自发性钙瞬变尚不能进行检测,及目前技术手段对ICCs细胞自发性钙瞬变观察的限制,不排除肌束内和具有不同功能的肌束旁ICCs细胞是膀胱起搏细胞的可能性;4.细胞外钙通过L型钙离子通道入胞在平滑肌细胞自发性钙瞬变发生中起主要作用,而细胞内钙库中的钙释放入胞浆可能起辅助作用;Ca²⁺通过T型钙离子通道入胞则可影响细胞自发性钙瞬变的发生频率;5.细胞外钙通过非L型钙离子通道入胞及胞内钙库中的钙释放均参与ICCs细胞自发性钙瞬变的发生;参与ICCs细胞自发性钙瞬变发生的细胞膜上的非L型钙离子通道可能是①钠钙交换体NCX、②T型钙离子通道和③TRP（瞬时受体势）离子通道中的多个通道;6. cADPR调节平滑肌细胞内质网钙释放的机制是:使FKBP12.6从RyR2上脱落下来,导致RyR2通道开放明显增强,内质网中钙离子经RyR2通道释放入胞浆显著增加。更多还原

【Abstract】 Bladder is a hollow muscular organ collects urine excreted by the kidneys prior to disposal by urination. The detrusor muscle is a layer of the urinary bladder wall made of smooth muscle fibers arranged in spiral, longitudinal, and circular bundles. As a speical smooth muscle tissue, the detrusor muscle is double controlled by splanchnic nerve and somatic nerves. Whether the detrusor muscle can generate spontaneous contraction like heart and gastrointestinal tract do was ever a mysterious question.Our previous research have demonstrated several interesting phenomenons:first, all the strips isolated from bladder exhibit spontaneous contraction activity under certain tension load, and the unstable strips will develop spontaneous contraction under little tension load; second, when under the same tension load, the frequency of unstable detrusor strips increases more significantly than that of normal detrusor; third, the spontaneous contraction is little affected by neural factors. These phenomenons indicated that the spontaneous contraction of detrusor strips may not be neurogenic, and the detrusor should have the ability of generating spontaneous excitability itself.It is also notable that the detrusor muscle exhibited significant increased activity of spontaneous contraction in a urological condition termed as DI（Detrusor instability） induced by bladder outlet obstruction or abnormal nerve function. Compared with normal detrusor strips, Smaller tension load is sufficient to induced spontaneous contractions in unstable detrusor strips.When under the same tension load, the frequency of unstable detrusor strips increases more significantly than that of normal detrusor strips.The enhanced spontaneous contraction also were observed in patients with overactive bladder （OAB）who suffered nearogenic bladder dysfunction and partial bladder outlet obstruction.The etiology of OAB is unclear, and indeed there may be multiple possible causes.In present,two different mechanism were made to explanation cause and development of OAB.① neurogenic mechanism:de Groat is one of supporter of neurogenic mechanism which emphasized that enhance spontaneous contraction all induced by abnormal nerve fuction（for example:up-regulation of number and function of neuroreceptors）and its subsequent effects.②Myogenci mechanism:Brading and other research insisted that besides neurogenic dysfunction, myogenic factors such as increased excitability of detrusor smooth muscle, enhaced communication between detusor smooth cells、ganglionic cells and up-regulation of the number and function of "pacemaker cells".As for myogenic mechanism of OAB, it is important for subsequent research to confirm whether pacemarker cells exist in bladder.ICCs（Interstitial cells of cajal） possesses different shape and function from smooth muscle cells.It has been accepted widely that as a pacemaker cell of gastrointestinal tract,ureter and other smooth muscle organs, spontaneous excitation of smooth muscle cells orginated from ICCs.With the help of immunofluorescence, ICCs（c-kit positive cells） were found distributed in bladder of guinea pig and human.ICCs, with Fusiform appearance, were showed to have a close relationship with smooth muscle cells and nerve ending and can generate spontaneous Ca²⁺ transients which suggested that ICCs may be the candidate of pacemaker cell of bladder. Subsequently,ICCs were found to drive spontaneous transient outward currents（STOCs） and Glivec, a specific c-kit inhibitot, were showed to significantly inbibit spontaneous action potentials（APs） of smooth muscle cells.These research data prompted that ICCs do have the ability of autodepolarization which confirmed the presence functionally and suggested that similiar gastrointestinal tract and ureter,ICCs may be the pacemaker cell in bladder.However, there are evidences points to the conclusion that APs can also be recorded in smooth muscle cells freshly isolated from bladder which prompted that smooth muscle cells may be another candidate of pacemaker cell of bladder spontaneous contraction .Then is ICCs or DSMCs the pacemaker cell of bladder? Research at tissue level should be taken to confirm since research data at single cell level are not sufficient to answer the question.Release of calcium from intracellular stores, endoplasmic/sarcoplasmic reticulum （ER/SR）, is one of the key signal transduction mechanisms that play a pivotal role in the regulation of numerous cellular functions. In smooth myocytes, Ca²⁺ release from intracellular Ca²⁺ stores to cytoplasm are controlled by ryanodine Ca²⁺ release receptors subtype 2 （RYR2）, which can occur in form of spontaneous SR Ca²⁺ release events, or Ca²⁺ sparks, and Ca²⁺ release that is triggered by the influx of Ca²⁺ through sarcolemmal ion channels, often termed CICR.Intracellular Ca²⁺-mediated excitation-contraction coupling in smooth muscle cells （DSMCs） is primarily controlled by the influx of extracellular Ca²⁺ and the mobilization of Ca²⁺ from intracellular stores. Ca²⁺ transients represent Ca²⁺ influx through voltage-dependent Ca²⁺ channels （VDCCs） during action potentials, the influx of extracellular Ca²⁺ produces smooth muscle constriction in part by stimulating Ca²⁺ release from the sarcoplasmic reticulum （SR） through Ca²⁺-induced Ca²⁺ release （CICR）.Cyclic adenosine diphosphate ribose （cADPR） is a naturally occurring cyclic nucleotide and represents a novel class of endogenous Ca²⁺ messengers implicated in the regulation of the gating properties of ryanodine receptors （RyRs）.In smooth muscle, cADPR can regulate Ca²⁺ transient by modulating Ca²⁺ release through RyR2 from SR. Alteration of gating properties of RyR2 induced by cADPR can regulate depolarization/repolarization and characteristic of Ca²⁺ transients of DSMCs by modulate STOCs（spontaneous transient outward current）, STICs（spontaneous transient outward currents） and CICR. Howerver,the role and mechanism of cADPR regulation of Ca²⁺ release remain unclear and very controversial and investigating of the mechanism will help us to further comprehend the procedure that cADPR modulate Ca²⁺ transients of DSMCs.Since there is a close relationship between Ca²⁺ transients and action potentials（APs） in smooth muscle tissue and cADPR is an important Ca²⁺ mobilizer which can modulate Ca²⁺ transients of DSMCs by regulating Ca²⁺ release from SR, we plan to do some works as following:First, immunofluorescence and immunofluorescence double staining protocol were used to obeserve the distribution of ICCs and its relationship with smooth muscle cells in the rat bladder;Second, spontaneous Ca²⁺ transients of ICCs and DSMCs were viewed in detrusor strips loaded with Fluo-4 AM by confocal microscopy, then amplitude and frequecy of spontanoues Ca²⁺ transients in ICCs and DSMCs were compared to initially investigate the function of ICCs;Third, a series of pharmacologic experiments were taken to explore the mechanism for generation and regulation of spontaneous Ca²⁺ transients in ICCs and DSMCs; finally, FKBP12.6 gene knockout mice were used to examine the effect and mechanism of cADPR on Ca²⁺ release in mouse bladder smooth muscle.Materials and methods: Female SD rats of 2-3 months,weighing from 220-280g were used. Immunofluorescence（anti-cKit）and immunofluorescence double staining （anti-ckit and anti-myosin） protocol were used to obeserve the distribution of ICCs and its relationship with smooth muscle cells in the rat bladder. By referencing to methods estabilished by hashitani,we recorded spontaneous Ca²⁺ transients of ICCs and smooth muscle cells with confocal microscopy.Serosa and mucosa removed bladder strips were prepared and superfused with warmed physiological saline（PSS） to recover and maintain their activity. After spontaneous muscle movements of the muscle layers were visually detected, the tissues were loaded with the fluo-4 AM and then spontaneous Ca²⁺ transients of ICCs in the muscle layers were observed with confocal microscopy. Then L type Ca²⁺ channel blocker Nimodipine, T type Ca²⁺ channel blockers Nicl2 ,Ca²⁺ free and High Ca²⁺ physiological saline and several reagents which can interfere SR Ca²⁺ release were added to investigate the mechanism of generation of spontaneous Ca²⁺ transients in ICCs and DSMCs.Then we sought to examine the effect and mechanism of cADPR on Ca²⁺ release in mouse bladder smooth muscle. We have used FKBP12.6 null mice to test directly if cADPR action on Ca²⁺ sparks is through FKBP12.6 disassociation from the RyR2 complex. Genetype of mice were tested by conducting a PCR （Polymerase Chain Reaction） on DNA samples taken from the mouse tail. Effects of cADPR on STOCs（spontaneous transient out currents） and Ca²⁺ sparks were recorded by patch clamp and confocal microscopy. Thapsigargin（A SERCA blocker） was used to detect whether SERCA activity is necessary for cADPR-induced increase in Ca²⁺ sparks.To further confirm that the effect of cADPR on Ca²⁺ spark properties is directly dependent on the dissociation of FKBP12.6 from the RyR2 complex, Western blotting analysis was performed on SR microsomes from mouse bladder smooth muscle. Previous studies demonstrated that the amount of FKBP12.6 bound to RYR2 can influence SR Ca²⁺ load. To determine whether cADPR affected SR Ca²⁺ content, caffeine （10 mM） was used to estimate SR Ca²⁺ load in myocytes of WT and FKBP12.6-null mice before and after cADPR treatment.Results:1. There are three categories ICCs in rat bladder strips, namely ICCs tracking smooth muscle boundary, ICCs between the bundles and ICCs in the bundles.There is a close relationship between ICCs and smooth muscle cells and neuro ending; 2. Properties of spontaneous Ca²⁺ transients recorded from DSMCs in detrusor strips（1） Asynchronous Ca²⁺ transients were obeserved right after the ending of Fluo-4 loading protocol.Some 30 min after superfusion with dye free physiological saline, synchronous Ca²⁺ transients generated in smooth muscle bundles;（2） Mostly, spontaneous Ca²⁺ transients of DSMCs originated along a boundary of each smooth muscle bundle and then spread to the other boundary.Some Ca²⁺ transients originated from the middle of the bundles and travelled to two boundaries.（3） Delay of propagation can be observed along the transverse direction but not along the axial direction;（4） Propagations of spontaneous Ca²⁺ transients between smooth muscle bundles were also observed by confocal laser scanning microscopy;3. Frequency of spontaneous Ca²⁺ transients recorded from DSMCs were significantly faster than frequency of that recorded from ICCs which suggested that ICCs tracking smooth muscle bundles may not be the pacemaker cell of bladder spontaneous contraction;4. Research data of subsequent pharmalogical experiments:（1） Nimodipine can abolish spontaneous Ca²⁺ transients in DSMCs. Frequency but not amplitude of spontaneous Ca²⁺ transients in DSMCs was significantly reduced by Nicl2. No alteration induced by Nimodipine and Nicl2 in spontaneous Ca²⁺ transient were observed in ICCs.（2） Ryanodien（RyR receptor antagonist）,Caffeine（RyR receptor agonist）,2-APB（IP3 receptor antagonist） and Thapsigargin（SERCA blocker） can significantly inhibite but can not abolish spontaneous Ca²⁺ transients of DSMCs.（3） Contrary to findings in DSMCs, spontaneous Ca²⁺ transients of ICCs can be abolished by Ryanodien,Caffeine,2-APBand Thapsigargin.（4） Ca²⁺ free physiological saline also are found can abolish spontaneous Ca²⁺ transients of ICCs.Frequncy of spontaneous Ca²⁺ transients of DSMCs can be increased significantly by high Ca²⁺ physiological solutions.5. Mice that are from heterozygous （one wildtype allele and one knockout） knockout parents were tested for their genotype by PCR. Three genetype were found in mice:Wildtype（FKBP12.6 +/+）,Knockout（FKBP12.6 -/-） and heterozygous （FKBP12.6 +/-）.6. On dialysis of 5μM cADPR into bladder smooth muscle cells via patch pipettes, both the frequency and amplitude of STOCs were significantly altered in wildtype cells.The frequency and amplitude of STOCs increased significantly in wildtype cells. In contrast to wildtype cells, the cells from FKBP12.6 null mice did not respond to cADPR stimulation.7. Ca²⁺ sparks are due to the concerted opening of multiple RyRs and STOCs were generated by Ca²⁺ Sparks.Patch clamp and confocal microscopy combined study showed that on dialysis of wildtype cells with cADPR, both STOCs and Ca²⁺ sparks were markedly altered. In contrast, dialysis of FKBP12.6 knockout cells with cADPR resulted in a negligible change in the properties of STOCs and Ca²⁺ sparks.8. After dialysis of cADPR, marked alterations were observed in WT cells but not in FKBP12.6 KO cells. Ca²⁺ spark frequency was increased significantly in wildtype but not in FKBP12.6 null cells. Similarly, on dialysis of wildtype cells with cADPR, peak Ca²⁺ （F/F0）,FWHM increased remarkably; rise timeand half-time decay were both prolonged markedly. However, cADPR did not alter Ca²⁺ spark properties in FKBP12.6 knockout cells.9. After dialysis of cADPR, Ca²⁺ spark frequency increased significantly and the alteration in Ca²⁺ spark frequency was not affected by thapsigargin. Similarly, increases in F/F0, FWHM, rise time and half-time decayinduced by cADPR were not markedly affected by thapsigargin.10. The endogenous FKBP12.6 was released from SR microsomes by incubation with cADPR and practically all of the FKBP12.6 was bound to SR microsomes in the pellet.11. It was notable that the FKBP12.6 protein deletion caused a reduction in Ca²⁺ content of SR in wildtype but not in FKBP12.6 null smooth muscle cells.Conclusion:1. Mechanism of spontaneous Ca²⁺ transients’ propogation along the transverse direction may be different from that along the axial direction and spontaneous Ca²⁺ transients’ propagate between smooth muscle bundles may have some relationship with etiology of detrusor instability;2. Asynchronous Ca²⁺ transients were transformed to synchronous Ca²⁺ transients by superfusion with dye free physiological saline in smooth muscle bundles suggested pacemaker cell did exist in bladder and candidate of the pacemaker may include:①DSMCs at the boundary of smooth bundles;②ICCs tracking smooth muscle boundary and ICCs in the bundles. However some Ca²⁺ transients also were found originated from the middle of the bundles suggested that different distribution of pacemaker cells may existed in distinct muscle bundles;3. Frequency of spontaneous Ca²⁺ transients recorded from DSMCs were significantly faster than that recorded from ICCs, which suggested that ICCs tracking smooth muscle bundles may not be the pacemaker cell of bladder spontaneous contraction. However, it is far from making a decision that ICCs tracking smooth muscle bundles are not the pacemake cell since we still have some technological problem（for example the limitation from focal length of confocal microscopy） and some ICCs tracking smooth muscle may have different functions. Besides, ICCs in the bundles may be another candidate of pacemaker cell of bladder sponteneous contraction;4. DSMCs generated spontaneous Ca²⁺ transients via extracellular Ca²⁺ entry through L type Ca²⁺ channels and the spontanoue Ca²⁺ transients were partly supported by Ca²⁺ release from Ca²⁺ release from intracellular Ca²⁺ stores; T-type Ca²⁺ channels may contribute to the preceding depolarization to activate the L-type Ca²⁺ channels and maintain the resting potential of DSMCs and thus determine the action potential frequency;5. spontaneous Ca²⁺ transients of ICCs rely on both Ca²⁺ release from intracellular Ca²⁺ stores and extracellular Ca²⁺ entry through non L type Ca²⁺ channels. Candidate of the non L type Ca²⁺ channels may include:①NCX(Na+/Ca²⁺ exchanger)②T type Ca²⁺ channels and③TRP channels;6. cADPR significantly alters Ca²⁺ spark properties and Ca²⁺ content in SR and that the underlying mechanism of cADPR action is related to the dissociation of FKBP12.6 proteins from the RYR2 complex in mouse bladder smooth muscle.更多还原