梗阻性DO逼尿肌细胞兴奋性变化及其离子通道机制研究
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摘要
背景及目的:逼尿肌过度活动(DO)是一种常见的膀胱功能障碍,可以引起尿急、尿频甚至急迫性尿失禁等临床表现,严重影响了患者的正常生活和身心健康,同时也日益成为社会公共医疗系统的沉重负担。正常膀胱在储尿期处于一种“安静”的状态,并且由于良好的顺应性,在一定容量范围内都保持很低的腔内压,这有利于尿液从上尿路的输送以及避免尿液的膀胱-输尿管反流。这个过程是下尿路器官在一系列复杂的神经、体液、心理意识以及自身等因素的调控下完成的协调过程。一旦由于某种原因,此种和谐遭到破坏,膀胱出现了不受意识控制的收缩,就会出现上述的临床表现,这些症候群被统称为膀胱过度活动症(overactive bladder,OAB),而这种自发性膀胱收缩则被称为逼尿肌过度活动(DO)并可以被临床尿动力学检查所记录和诊断。DO在西方国家发病率很高,被列为10种最常见的慢性疾病之一;国内虽然没有大规模的临床流行病学报告确切的发病率,然而就泌尿临床诊治情况来看,属于非常常见的疾病。
DO目前尚无理想的治疗方法,最常用抗胆碱能药物进行治疗,但由于副作用过大,疗效有限,患者的治疗依从性非常差。因此,迫切需要寻找新的有效的替代疗法。关于DO的发病机制已经进行了大量的基础研究。综合来看,原因复杂,可能系多因素参与致病,不同病因导致的DO可能有不同的发病机理,但确切的发病机制还远未明了。目前,主要学说可归纳成神经源性和肌源性两种。按照ICS的分类,DO可分成特发性(idiopathic detrusor overactivity)和神经源性(neurogenic detrusor overactivity)两种。具有相关联的神经系统病变者称为神经源性,其余归为特发性,而后者最常继发于BOO性疾病,其中以BPH最为多见。尽管DO可能是多因素疾病,但目前已有大量证据表明,继发于梗阻后的膀胱逼尿肌本身结构和功能的改变在BOO所致DO的发生中占有极为重要的地位,即“肌源性”机理。针对不同病因所致DO可能具有不同发病机理这一特点,并且考虑到BOO所致DO为临床最为常见的类型,本课题拟针对BOO性DO的发生机理展开研究。在“肌源性”学说所提出的重要机理方面,认为“逼尿肌兴奋性升高与DO有着密切的关系”。但我们广泛查阅了相关文献后发现,这一重要的机理尚缺乏直接的证据支持。为此,以此做为课题的切入点。首先,第一部分应用目前研究细胞兴奋性最好的技术-穿孔膜片钳全细胞记录,在具有“真电流钳”功能的新型膜片钳放大器EPC-10上,选取评价兴奋性的可靠指标,对BOO性DO大鼠逼尿的兴奋性与正常逼尿肌进行检测与比较。然后,进一步研究这种高兴奋状态的具体分子机理(离子通道机制),选取在调控正常逼尿肌兴奋性中具有最重要作用的电压依赖性钙通道(VDCC)和大电导钙激活钾通道(BKca)作为主要研究对象,对逼尿肌兴奋的启动和抑制这两个层面进行考察。沿着这条主线,在第二部分中主要针对VDCC中的L型和T型的变化进行探索。综合应用膜片钳技术、激光共聚焦逼尿肌钙荧光探针浓度测量并结合相应的药理学工具,对BOO性DO逼尿肌VDCC中的L和T性钙通道的电学特性及其在细胞静息钙浓度调控进行检测并与正常逼尿肌进行比较,明确钙通道活动的改变及其与高兴奋性的关系;在第三部分,应用膜片钳技术,从单通道、全细胞电流密度以及瞬时外向钾电流(STOCs)三个方面对BKca通道的活性改变进行观察和比较,同时应用免疫印迹技术对BKca通道α、β亚单位的蛋白表达改变进行比较,明确BKca蛋白表达和功能活动的改变以及与高兴奋状态的关系。
材料和方法
1.采用近端尿道不全结扎法制作PBOO性雌性大鼠DO动物模型;
2.模型制作后6-8周应用尿流动力仪充盈性膀胱测压筛选DO模型和对照。出现膀胱非排尿性收缩的尿道结扎大鼠作为DO组,无非排尿性收缩表现的正常同龄大鼠作为对照组;
3.采用消化酶法急性分离适合膜片钳技术的单个逼尿肌细胞;
4.用以两性霉素B为穿孔剂的穿孔膜片钳电流钳下记录逼尿肌细胞的静息电位、输入膜阻抗、阈刺激、动作电位阈电位等指标;
5.用穿孔膜片钳全细胞电压钳下记录逼尿肌L和T型钙通道的峰值电流密度以及激活曲线、稳态失活曲线,及药理学特性;
6.观察L和T型钙通道阻断剂对Fluo-4AM钙荧光探针标记的逼尿肌细胞静息钙浓度的影响;
7.在穿孔膜片钳全细胞电压钳模式下记录BKca全细胞电流和STOCs电流,分析全细胞电流密度,STOCs平均电流幅度及平均发放频率。
8.应用inside-out膜片记录逼尿肌BKca单通道电流,并对单通道特性进行电学和药理学鉴定,分析开放概率、平均开放时间、平均关闭时间、电导等参数;
9.应用western blot技术对BKcaα、β亚单位蛋白表达情况进行比较。
结果:
(一)成功建立了大鼠BOO性DO动物模型,模型成功率高达82.3%。
(二)成功建立了一种逼尿肌细胞急性酶分离方法,具有重复性好、操作简单的优点,并且适于膜片钳研究。
(三)成功地将穿孔膜片钳技术技术应用在大鼠逼尿肌膜电位记录上,最大程度地反应了逼尿肌的电生理特性。
(四)对DO和正常逼尿肌细胞兴奋性进行了各预设指标的检查,结果分析表明,BOO性DO逼尿肌细胞兴奋性较正常显著增加,这可能是DO发生的重要原因。
(五)首次成功地进行了逼尿肌细胞的穿孔膜片钳电压依赖性钙通道电流的记录
(六) DO逼尿肌L型钙通道发生变化,表现为:
1.通道的密度显著增加,激活后可引起更多的钙内流,这可能是DO后逼尿肌收缩力增强的原因之一。
2.通道失活曲线右移,处于失活状态的L型钙通道比例减少,提示:L型钙通道阻断剂需要使用更大的浓度才能将其完全抑制。
(七) DO逼尿肌T型钙通道发生变化,表现为:
1.通道的密度显著增加。
2.通道失活曲线右移,激活曲线左移使得静息状态下T通道的“window current”(窗电流)增大,这是DO逼尿肌静息钙浓度增加的重要原因之一;
3.通道激活曲线左移,这可能是DO兴奋性增加的重要原因。
4.T型钙通道对于正常和DO逼尿肌中的静息钙浓度均有显著的调控作用,这是通过其窗电流实现的。使用T型钙通道阻断剂后,钙荧光的前后变化率DO逼尿肌大于正常逼尿肌,证明T型钙通道形成窗电流在DO逼尿肌是上调的。
(八) DO逼尿肌BKca全细胞电流密度显著下降。
(九) DO逼尿肌BKca单通道活动显著下降,具体为开放概率、平均开放时间下降而平均关闭时间以及单通道电导未发生改变。同时,单通道对钙敏感性下降。
(十) DO逼尿肌瞬时外向钾电流(STOCs)的幅度以及频率显著下降.。
(十一) DO逼尿肌BKca通道α、β亚单位蛋白表达显著下降.
结论
一、梗阻性DO逼尿肌细胞兴奋性显著高于正常逼尿肌,这可能是BOO后发生DO的重要原因。
二、DO逼尿肌细胞L和T性钙通道密度增加,动力学发生诸多改变。这些改变易化了DO逼尿肌产生动作电位的能力,是其兴奋性升高的重要离子基础。
三、DO逼尿肌中BKCa通道的蛋白表达和通道活动均出现显著的削弱,这使得其抑制细胞兴奋的能力严重减弱,这些变化也是DO逼尿肌兴奋性升高重要的离子基础。
Background and Objective
Detrusor overactivity (DO) is a very common urinary dysfunction which could give rise to a variety of irritating symptoms such as urgency ,frequency with or without urge incontinence .DO could seriously affect the suffers’life quality or even mental health and it now apparently becomes a heavy burden to the social medical system.When in the filling stage,the healthy urinary bladder is kept in“quiescence”and low intravesical pressure is maintained due to the excellent compliance of the urinary bladder which is of help to the urine transmission from the upper urinary tract to the bladder and in preventing vesico-ureteric reflux .This physiological process is under complex control of central nervous system,body humour and consciousness along with the low urinary organ themselves.However,whatever reason,if the above harmonious interactions are interrupted ,the urinary bladder could exhibit uninhibited involuntary contractions during the filling stage which in turn induces the above mentioned symptoms complex.This symptoms complex is termed as the overactive bladder syndrome (OAB) ,whilst the involuntary urinary bladder contraction is referred to as detrusor overactivity (DO) which could be recorded and diagnosed by a cystometry assay.In accordance with the latest clinical epidemiology report,the incidence of DO is very high and it is among the top 10 most common chronic diseases in western world .Though the corresponding investigation is lacking regarding Chinese population ,it is however indeed occupy a big portion of urological clinic workload suggesting similarly high incidentce in China. Unfortunately,there is few effective therapeutic means available to treat DO patients .The anticholinergic agents are now the first line drug,however,because of their seriously unpleasant side-effects such as dry mouth and constipation ,also mild and limited effectiveness in relieving OAB symptoms,few patients can comply with this drug for long period of time,therefore , more effective strategies with less side effects must be come up with in a short time in order to deal with this trying situation.
Though a lot have been done regarding the pathophysiology of DO,unfortunately, it is still largely unknown.The currnt opinions are that DO is caused by multifaceted etiologies and the responsible mechanisms might vary among DO with distinctive origins but the only thing with certainty is that the exact pathological mechanism is not uncovered yet.If they could be summed up ,the mainstay theories can be attributable to two main categories ,ie,one is the neurogenic displine and the other is myogenic one with strikingly contrasting arguments stressing on nerve drive or musle drive respectively.The newest definition about DO from the authoritative ICS is that this disease entity could follow into two categories.One is the neurogenic DO if the suffers are demonstrated with a related neurological impairment and however,if not it is called the idiopathetic DO which is commonly secondary to BOO with the BPH as the most frequent reason. Accumulating evidence has now indicate that the morphological and functional detrusor remolding after BOO play a pivotal role in the DO’s pathophysiology .Given that DO with distinctive etiology may vary in pathological mechanisms and the DO caused by BOO is the most common type ,we decided to choose the DO arising from BOO as our working model. According to myogenic displine of DO,the hyperexcitability of detrusor myocytes is closely associated with DO but however,the direct evidence is lacking after our painstaking literature reviewing,so it is reasonable to choose this myth as our focus because this is an extremely important issue and might bear fruitful results by resolving it.Our study has 3 parts.In the first part ,we firstly establish the PBOO rat model and then measured the cellular excitability index by the aid of the perforated patch clamp recording which is the best one in the physiologists’disposal when addressing the excitability investigation.In the second part,we investigate the L and T type VDCC channels change by using a combined methods,ie.the perforated whole cell patch clamp and calcium concentration measurement under LSCM using the fluo-4AM calcium sensitive fluorescence dye in the hope to reflect the VDCC channel activities change in DO detrusor myocytes from different perspectives.In the last part,the BKca channel activity changes were measured by patch clamp technique and its protein expression changes were assayed by western blot .
Materials and methods
1. The PBOO animal model was established by proximal urethral ligation in the female SD rats.
2. Between the sixth and eighth week after animal model ,the validation of DO was performed by a cystometry assay and based on results of this investigation,the rats were grouped as DO and control(healthy rat with matching age but without DO)
3. The detrusor myocytes were dispersed by enzymatic digestion.
4. The resting membrane potential ,input resistance ,action potential voltage etc were measured by the whole cell perforated patch clamp configuration under the current clamp mode.
5. The L and T type VDCC currents were recorded by perforated patch clamp and peak current density and channel kinetic curve were obtained .
6. The modulation roles of L and T VDCC on the resting detrusor free calcium concentration were probed by the VDCC antagonists through LCSM calcium concentration measurement method.
7. The whole cell BKca currents and STOCs were recorded by perforated patch clamp.
8. The single BKca channel activities were measured by patch clamp under inside-out patch configuration.
9. The protein level of BKcaα、βsubunits were assayed by western blot .respectively.
Results
1.The DO rat model induced by PBOO is successfully established and the incidence is as high as 82.3%.
2 . A simple and highly reproducible detrusor myocytes dispersion method is successfully established and they are proved to be suitable for patch clamp study.
3.The perforated patch clamp configuration which is the most physiological method for cellular electrophysiological study is successfully applied in recording the single detrusor membrane potential
4.The results by comparing excitability index between DO and normal detrusor myocytes indicate that the DO detrusor myocytes have much higher excitability than the normal control ,which is a possible reason for DO .
5.The first perforated patch recording of voltage gated calcium currents on detrusor myocytes is successfully done .
6.The L type calcium channels have changed in DO myocytes as follows
(1) The current density increase significantly which will result in more calcium influx after activation
(2) The inactivation curve shifts to the depolarization direction and the percentage of channels in inactivating state decrease implying that it needs much higher concentration of calcium channel blockers to completely inhibit the channel opening .
7.The T type calcium channels have changed in DO myocytes as follows
(1) The current density increase significantly .
(2) The window currents of the T channels widen as a result of shift of both activation and inactivation curve.,which would increase the resting free calcium concentration.
(3) The activation curve shifts to the hyperpolarization direction which might underly the hyperexcitability of DO detrusor myocytes.
(4) T type calcium channels play a vital role in modulating the resting free calcium concentration of both normal and DO detrusor myocytes through the window currents pathway.The change ratio of RCFI is higher in DO detrusor than that in normal after adding the T channel antagonist mibefradil suggesting that the window currents are up-regulated in DO state .
8.The whole cell BKca current density decrease in DO detrusor.
9.The single channel activities also decrease in DO detrusor myocytes significantly manifested by decrease of both open probability and mean open time.
10.Both the frequency and amplitude of detrusor STOCs decrease significantly.
11.The protein expression level of bothαandβsubunites of BKca channels decreases significantly.
Conclusions
1.The detrusor from DO model exhibit hyperexcitability which maybe a key reason for DO .
2.Both the L and T type calcium channels have profound changes such as increase of current density and shift of kinetic curve which will facilitate the action potential firing and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability.
3.Both the protein expression and channel activities of BKca channels decrease significantly in DO detrusor which will impair its capacity to antagonize the excitability and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability.
DO目前尚无理想的治疗方法,最常用抗胆碱能药物进行治疗,但由于副作用过大,疗效有限,患者的治疗依从性非常差。因此,迫切需要寻找新的有效的替代疗法。关于DO的发病机制已经进行了大量的基础研究。综合来看,原因复杂,可能系多因素参与致病,不同病因导致的DO可能有不同的发病机理,但确切的发病机制还远未明了。目前,主要学说可归纳成神经源性和肌源性两种。按照ICS的分类,DO可分成特发性(idiopathic detrusor overactivity)和神经源性(neurogenic detrusor overactivity)两种。具有相关联的神经系统病变者称为神经源性,其余归为特发性,而后者最常继发于BOO性疾病,其中以BPH最为多见。尽管DO可能是多因素疾病,但目前已有大量证据表明,继发于梗阻后的膀胱逼尿肌本身结构和功能的改变在BOO所致DO的发生中占有极为重要的地位,即“肌源性”机理。针对不同病因所致DO可能具有不同发病机理这一特点,并且考虑到BOO所致DO为临床最为常见的类型,本课题拟针对BOO性DO的发生机理展开研究。在“肌源性”学说所提出的重要机理方面,认为“逼尿肌兴奋性升高与DO有着密切的关系”。但我们广泛查阅了相关文献后发现,这一重要的机理尚缺乏直接的证据支持。为此,以此做为课题的切入点。首先,第一部分应用目前研究细胞兴奋性最好的技术-穿孔膜片钳全细胞记录,在具有“真电流钳”功能的新型膜片钳放大器EPC-10上,选取评价兴奋性的可靠指标,对BOO性DO大鼠逼尿的兴奋性与正常逼尿肌进行检测与比较。然后,进一步研究这种高兴奋状态的具体分子机理(离子通道机制),选取在调控正常逼尿肌兴奋性中具有最重要作用的电压依赖性钙通道(VDCC)和大电导钙激活钾通道(BKca)作为主要研究对象,对逼尿肌兴奋的启动和抑制这两个层面进行考察。沿着这条主线,在第二部分中主要针对VDCC中的L型和T型的变化进行探索。综合应用膜片钳技术、激光共聚焦逼尿肌钙荧光探针浓度测量并结合相应的药理学工具,对BOO性DO逼尿肌VDCC中的L和T性钙通道的电学特性及其在细胞静息钙浓度调控进行检测并与正常逼尿肌进行比较,明确钙通道活动的改变及其与高兴奋性的关系;在第三部分,应用膜片钳技术,从单通道、全细胞电流密度以及瞬时外向钾电流(STOCs)三个方面对BKca通道的活性改变进行观察和比较,同时应用免疫印迹技术对BKca通道α、β亚单位的蛋白表达改变进行比较,明确BKca蛋白表达和功能活动的改变以及与高兴奋状态的关系。
材料和方法
1.采用近端尿道不全结扎法制作PBOO性雌性大鼠DO动物模型;
2.模型制作后6-8周应用尿流动力仪充盈性膀胱测压筛选DO模型和对照。出现膀胱非排尿性收缩的尿道结扎大鼠作为DO组,无非排尿性收缩表现的正常同龄大鼠作为对照组;
3.采用消化酶法急性分离适合膜片钳技术的单个逼尿肌细胞;
4.用以两性霉素B为穿孔剂的穿孔膜片钳电流钳下记录逼尿肌细胞的静息电位、输入膜阻抗、阈刺激、动作电位阈电位等指标;
5.用穿孔膜片钳全细胞电压钳下记录逼尿肌L和T型钙通道的峰值电流密度以及激活曲线、稳态失活曲线,及药理学特性;
6.观察L和T型钙通道阻断剂对Fluo-4AM钙荧光探针标记的逼尿肌细胞静息钙浓度的影响;
7.在穿孔膜片钳全细胞电压钳模式下记录BKca全细胞电流和STOCs电流,分析全细胞电流密度,STOCs平均电流幅度及平均发放频率。
8.应用inside-out膜片记录逼尿肌BKca单通道电流,并对单通道特性进行电学和药理学鉴定,分析开放概率、平均开放时间、平均关闭时间、电导等参数;
9.应用western blot技术对BKcaα、β亚单位蛋白表达情况进行比较。
结果:
(一)成功建立了大鼠BOO性DO动物模型,模型成功率高达82.3%。
(二)成功建立了一种逼尿肌细胞急性酶分离方法,具有重复性好、操作简单的优点,并且适于膜片钳研究。
(三)成功地将穿孔膜片钳技术技术应用在大鼠逼尿肌膜电位记录上,最大程度地反应了逼尿肌的电生理特性。
(四)对DO和正常逼尿肌细胞兴奋性进行了各预设指标的检查,结果分析表明,BOO性DO逼尿肌细胞兴奋性较正常显著增加,这可能是DO发生的重要原因。
(五)首次成功地进行了逼尿肌细胞的穿孔膜片钳电压依赖性钙通道电流的记录
(六) DO逼尿肌L型钙通道发生变化,表现为:
1.通道的密度显著增加,激活后可引起更多的钙内流,这可能是DO后逼尿肌收缩力增强的原因之一。
2.通道失活曲线右移,处于失活状态的L型钙通道比例减少,提示:L型钙通道阻断剂需要使用更大的浓度才能将其完全抑制。
(七) DO逼尿肌T型钙通道发生变化,表现为:
1.通道的密度显著增加。
2.通道失活曲线右移,激活曲线左移使得静息状态下T通道的“window current”(窗电流)增大,这是DO逼尿肌静息钙浓度增加的重要原因之一;
3.通道激活曲线左移,这可能是DO兴奋性增加的重要原因。
4.T型钙通道对于正常和DO逼尿肌中的静息钙浓度均有显著的调控作用,这是通过其窗电流实现的。使用T型钙通道阻断剂后,钙荧光的前后变化率DO逼尿肌大于正常逼尿肌,证明T型钙通道形成窗电流在DO逼尿肌是上调的。
(八) DO逼尿肌BKca全细胞电流密度显著下降。
(九) DO逼尿肌BKca单通道活动显著下降,具体为开放概率、平均开放时间下降而平均关闭时间以及单通道电导未发生改变。同时,单通道对钙敏感性下降。
(十) DO逼尿肌瞬时外向钾电流(STOCs)的幅度以及频率显著下降.。
(十一) DO逼尿肌BKca通道α、β亚单位蛋白表达显著下降.
结论
一、梗阻性DO逼尿肌细胞兴奋性显著高于正常逼尿肌,这可能是BOO后发生DO的重要原因。
二、DO逼尿肌细胞L和T性钙通道密度增加,动力学发生诸多改变。这些改变易化了DO逼尿肌产生动作电位的能力,是其兴奋性升高的重要离子基础。
三、DO逼尿肌中BKCa通道的蛋白表达和通道活动均出现显著的削弱,这使得其抑制细胞兴奋的能力严重减弱,这些变化也是DO逼尿肌兴奋性升高重要的离子基础。
Background and Objective
Detrusor overactivity (DO) is a very common urinary dysfunction which could give rise to a variety of irritating symptoms such as urgency ,frequency with or without urge incontinence .DO could seriously affect the suffers’life quality or even mental health and it now apparently becomes a heavy burden to the social medical system.When in the filling stage,the healthy urinary bladder is kept in“quiescence”and low intravesical pressure is maintained due to the excellent compliance of the urinary bladder which is of help to the urine transmission from the upper urinary tract to the bladder and in preventing vesico-ureteric reflux .This physiological process is under complex control of central nervous system,body humour and consciousness along with the low urinary organ themselves.However,whatever reason,if the above harmonious interactions are interrupted ,the urinary bladder could exhibit uninhibited involuntary contractions during the filling stage which in turn induces the above mentioned symptoms complex.This symptoms complex is termed as the overactive bladder syndrome (OAB) ,whilst the involuntary urinary bladder contraction is referred to as detrusor overactivity (DO) which could be recorded and diagnosed by a cystometry assay.In accordance with the latest clinical epidemiology report,the incidence of DO is very high and it is among the top 10 most common chronic diseases in western world .Though the corresponding investigation is lacking regarding Chinese population ,it is however indeed occupy a big portion of urological clinic workload suggesting similarly high incidentce in China. Unfortunately,there is few effective therapeutic means available to treat DO patients .The anticholinergic agents are now the first line drug,however,because of their seriously unpleasant side-effects such as dry mouth and constipation ,also mild and limited effectiveness in relieving OAB symptoms,few patients can comply with this drug for long period of time,therefore , more effective strategies with less side effects must be come up with in a short time in order to deal with this trying situation.
Though a lot have been done regarding the pathophysiology of DO,unfortunately, it is still largely unknown.The currnt opinions are that DO is caused by multifaceted etiologies and the responsible mechanisms might vary among DO with distinctive origins but the only thing with certainty is that the exact pathological mechanism is not uncovered yet.If they could be summed up ,the mainstay theories can be attributable to two main categories ,ie,one is the neurogenic displine and the other is myogenic one with strikingly contrasting arguments stressing on nerve drive or musle drive respectively.The newest definition about DO from the authoritative ICS is that this disease entity could follow into two categories.One is the neurogenic DO if the suffers are demonstrated with a related neurological impairment and however,if not it is called the idiopathetic DO which is commonly secondary to BOO with the BPH as the most frequent reason. Accumulating evidence has now indicate that the morphological and functional detrusor remolding after BOO play a pivotal role in the DO’s pathophysiology .Given that DO with distinctive etiology may vary in pathological mechanisms and the DO caused by BOO is the most common type ,we decided to choose the DO arising from BOO as our working model. According to myogenic displine of DO,the hyperexcitability of detrusor myocytes is closely associated with DO but however,the direct evidence is lacking after our painstaking literature reviewing,so it is reasonable to choose this myth as our focus because this is an extremely important issue and might bear fruitful results by resolving it.Our study has 3 parts.In the first part ,we firstly establish the PBOO rat model and then measured the cellular excitability index by the aid of the perforated patch clamp recording which is the best one in the physiologists’disposal when addressing the excitability investigation.In the second part,we investigate the L and T type VDCC channels change by using a combined methods,ie.the perforated whole cell patch clamp and calcium concentration measurement under LSCM using the fluo-4AM calcium sensitive fluorescence dye in the hope to reflect the VDCC channel activities change in DO detrusor myocytes from different perspectives.In the last part,the BKca channel activity changes were measured by patch clamp technique and its protein expression changes were assayed by western blot .
Materials and methods
1. The PBOO animal model was established by proximal urethral ligation in the female SD rats.
2. Between the sixth and eighth week after animal model ,the validation of DO was performed by a cystometry assay and based on results of this investigation,the rats were grouped as DO and control(healthy rat with matching age but without DO)
3. The detrusor myocytes were dispersed by enzymatic digestion.
4. The resting membrane potential ,input resistance ,action potential voltage etc were measured by the whole cell perforated patch clamp configuration under the current clamp mode.
5. The L and T type VDCC currents were recorded by perforated patch clamp and peak current density and channel kinetic curve were obtained .
6. The modulation roles of L and T VDCC on the resting detrusor free calcium concentration were probed by the VDCC antagonists through LCSM calcium concentration measurement method.
7. The whole cell BKca currents and STOCs were recorded by perforated patch clamp.
8. The single BKca channel activities were measured by patch clamp under inside-out patch configuration.
9. The protein level of BKcaα、βsubunits were assayed by western blot .respectively.
Results
1.The DO rat model induced by PBOO is successfully established and the incidence is as high as 82.3%.
2 . A simple and highly reproducible detrusor myocytes dispersion method is successfully established and they are proved to be suitable for patch clamp study.
3.The perforated patch clamp configuration which is the most physiological method for cellular electrophysiological study is successfully applied in recording the single detrusor membrane potential
4.The results by comparing excitability index between DO and normal detrusor myocytes indicate that the DO detrusor myocytes have much higher excitability than the normal control ,which is a possible reason for DO .
5.The first perforated patch recording of voltage gated calcium currents on detrusor myocytes is successfully done .
6.The L type calcium channels have changed in DO myocytes as follows
(1) The current density increase significantly which will result in more calcium influx after activation
(2) The inactivation curve shifts to the depolarization direction and the percentage of channels in inactivating state decrease implying that it needs much higher concentration of calcium channel blockers to completely inhibit the channel opening .
7.The T type calcium channels have changed in DO myocytes as follows
(1) The current density increase significantly .
(2) The window currents of the T channels widen as a result of shift of both activation and inactivation curve.,which would increase the resting free calcium concentration.
(3) The activation curve shifts to the hyperpolarization direction which might underly the hyperexcitability of DO detrusor myocytes.
(4) T type calcium channels play a vital role in modulating the resting free calcium concentration of both normal and DO detrusor myocytes through the window currents pathway.The change ratio of RCFI is higher in DO detrusor than that in normal after adding the T channel antagonist mibefradil suggesting that the window currents are up-regulated in DO state .
8.The whole cell BKca current density decrease in DO detrusor.
9.The single channel activities also decrease in DO detrusor myocytes significantly manifested by decrease of both open probability and mean open time.
10.Both the frequency and amplitude of detrusor STOCs decrease significantly.
11.The protein expression level of bothαandβsubunites of BKca channels decreases significantly.
Conclusions
1.The detrusor from DO model exhibit hyperexcitability which maybe a key reason for DO .
2.Both the L and T type calcium channels have profound changes such as increase of current density and shift of kinetic curve which will facilitate the action potential firing and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability.
3.Both the protein expression and channel activities of BKca channels decrease significantly in DO detrusor which will impair its capacity to antagonize the excitability and may be one of the important ionic mechanisms underlying DO detrusor hyperexcitability.
引文
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