多巴胺受体在人食管下括约肌的表达及功能研究
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摘要
人食管下括约肌(Lower esophageal sphincter, LES)是位于食管胃结合部（Esophageogastric junction, EGJ）约2-3cm宽的特殊增厚环形肌。一般认为人LES是由胃小弯侧的半环形钩状纤维和大弯侧的斜行套索纤维所构成的。这两束肌纤维与膈肌脚一起，在LES肌源性和神经源性的因素作用下保持持续性收缩的状态，最终在EGJ形成高压带，从而防止胃内容物反流入食管；而另一方面，当发生吞咽和食管扩张时，在迷走神经的抑制性通路的作用下可以引起一过性的LES舒张（Transient lower oesophagealsphincter relaxation, TLESR），TLESR的存在，允许了食物继续下行入胃，或形成呕吐、打嗝，从而使胃内的液体或气体反流进入食管。
LES的收缩和舒张的功能调节机制是在中枢神经系统的支配下，由多种激素、神经递质及自身肌源性因素共同参与完成的。一般认为迷走神经输出神经末梢与LES肌层肠道运动神经元（Enteric Motor Neurons, EMN）形成突触联系，共同构成了调节LES收缩和舒张的兴奋性迷走神经通路和抑制性迷走神经通路。兴奋性迷走神经通路由节前胆碱能神经元和节后胆碱能神经元共同构成。该通路的激活可以引起LES收缩。而抑制性迷走神经通路则是由节前胆碱能神经元和节后非肾上腺非胆碱能（NonadrenergicNoncholinergic, NANC）神经元构成。激活抑制性通路可以引起LES舒张。
多巴胺（Dopamine, DA）是儿茶酚胺类神经递质之一，在自主运动、认知、情感、激素调节以及胃肠运动等功能的调节中发挥着重要的作用。这些功能的调节均是通过多巴胺受体（Dopamine receptor, DAR）实现的。DAR作为含有七跨膜结构域G蛋白偶联受体家族重要的受体类型之一，广泛存在于哺乳动物的中枢及外周神经系统。到目前为止已经发现DAR共有D1R-D5R五种亚型，按照各自的结构，药理学性质以及生物学属性可以将这些受体亚型分为D1样受体（D1R、D5R）和D2样受体（D2R、D3R、D4R）。近些年的研究表明，DAR广泛地分布于动物的胃肠道之中。
多种食管运动功能异常性疾病如贲门失弛缓、弥漫性食管痉挛、胡桃夹食管等均与LES的运动异常有关，目前研究表明LES的调节机制涉及多种受体，神经递质和信号转导通路。研究表明胆囊收缩素受体（Cholecystokinin receptor），毒蕈碱受体（Muscarinic receptor）均在LES的调节机制中发挥着重要的作用。
本研究采用逆转录聚合酶链反应(RT-PCR)，蛋白印迹法（Western-blot），离体肌张力测定技术以及电场刺激（Electrical fieldstimulation, EFS）等方法对DAR在人LES中的表达及功能进行研究。探讨了DAR在人LES调节机制中的作用，为进一步研究DA及其受体在人LES调节机制中的作用奠定基础，从而更为完善的阐述人LES调节机制，为食管运动功能障碍性疾病的临床治疗提供理论依据。
第一部分多巴胺受体在人食管下括约肌的表达研究目的：DAR是G蛋白偶联受体家族重要的受体类型之一，介导着儿茶酚胺类神经递质DA所有的生理学功能。本试验采用逆转录聚合酶链反应(RT-PCR)和蛋白印迹法（Western-blot）研究人LES的DAR的5种亚型在钩状纤维、套索纤维、食管和胃底的环行肌中mRNA、受体蛋白的表达规律，探讨DAR在人LES的功能调节中发挥的作用。
方法：选取2007年12月至2008年10月在河北医科大学第四医院因高位食管中段癌，而行食管大部切除术的患者共30例。其中23名男性，7名女性，平均年龄64岁。手术室收集新鲜EGJ手术标本，实验室锐性剥去食管下段、贲门及胃底的粘膜及粘膜下层后，制备钩状纤维、套索纤维、食管和胃底环行肌的肌条。提取组织RNA，经紫外分光光度计和1％琼脂糖凝胶电泳鉴定其纯度和完整性之后，分别应用5种DAR亚型的引物行RT-PCR，检测其mRNA在4种肌条的表达，用Gel Pro软件分析扩增产物的光密度值(IOD)，并计算DAR亚型与β-actin IOD的比值表示肌条中mRNA的相对含量。提取组织蛋白，经考马斯亮蓝G-250定量，将蛋白调整至相同浓度。然后应用电泳仪电泳，分离出与DAR各个亚型相应的受体蛋白后转膜，转膜结束后分别应用DAR各个亚型的抗体进行孵育及DAB染色，经Gel Pro软件分析DAB反应条带的光密度值(IOD)。
结果：紫外分光度计测定显示，总RNAA260/A280比值为1.6-1.8；1%琼脂糖凝胶电泳可见28S条带的宽度和亮度均是18S条带的两倍。内参β-actin于838bp处亮度均匀，在各个反应中，内参β-actin的PCR扩增结果一致。3种DAR亚型的mRNA在四种肌条中均可见表达，分别是D_1R，D_2R，D_5R。D_3R，D_4R的mRNA在四种肌条内均未见表达。PCR扩增产物的长度与其设计长度完全一致。同一肌条不同DAR亚型的mRNA表达水平的比较有统计学差异(F=669.00, P=0.00)，其表达水平的强度依次为：D1R＞D5R＞D2R。同一DAR亚型的mRNA表达水平在不同的肌条间的比较没有统计学差异(F=0.18, P=0.90)。有3种DAR的受体亚型的蛋白在四种肌条内均可见表达，分别是D1R，D2R，D5R，其分子量大小依次为51KD、51KD、53KD。D3R，D4R的蛋白在四种肌条内均未见表达。同一肌条不同DAR亚型的蛋白表达水平的比较有统计学差异(F=84.53, P=0.00)，其蛋白表达水平的强弱与mRNA表达水平的结果一致。同一DAR亚型的蛋白表达水平在不同的肌条间比较没有统计学差异(F=0.10, P=0.96)。
结论：人LES存在3种DAR亚型，分别是D1R，D2R，D5R。其表达水平的高低依次为D1R>D5R> D2R，可能在LES的功能调节中发挥着作用。D3R和D4R在人LES中无表达，可能对LES的功能调节没有影响。
第二部分多巴胺受体在人食管下括约肌调节机制中的功能研究
目的：研究非选择性DAR的激动剂和拮抗剂以及选择性的DAR的激动剂对人离体LES的钩状纤维（clasp）、套索纤维（sling）的作用，探讨DAR在人LES收缩和舒张的调节机制中所发挥的的作用。
方法：选取2010年8月到2011年3月在河北医科大学第四医院因高位食管中段癌行食管大部切除术患者30例。男性患者17例，女性患者13例，平均年龄62岁。手术室采集新鲜标本，于标记处沿胃大弯侧切开标本，粘膜面向上固定于盛有Krebs液的蜡盘中，持续通以95％O2和5％CO_2的混合气体。锐性剥离胃贲门部以及食管下段的粘膜层及粘膜下层，可见食管胃结合部的发白增厚的肌肉环，即为LES。肉眼识别出胃小弯侧位于标本的中央呈半环形的钩状纤维（clasp）与胃大弯侧位于标本的两端并呈斜行的套索纤维（sling）。沿着各自肌纤维走行的方向锐性游离钩状纤维（clasp）和套索纤维（sling），制备2mm×10mm的肌条。应用丝线分别将肌条的两端扎紧，置于含Krebs液10ml的浴槽中，保持37℃恒温条件。持续通以95%O2和5%CO2的混合气体。肌条下端固定，上端固定于JZ101型肌肉张力换能器，经Medlab信号采集系统记录张力变化情况。轻微缓慢牵拉肌条使之张力维持于200mg，此时的肌条长度作为初始长度L0，然后多次缓慢牵拉肌条，每次增加其初始长度L0的25％左右，每次待被动张力稳定15min后，再次牵拉。如此反复，每次牵拉间隔约20min，直至牵拉肌条长度至初始长度L0的200％为最适初长度。待肌条最适初长度稳定约40分钟后，以浓度累积方式向浴槽中加入非选择性DAR激动剂（Dopamine hydrochloride, DA），给药浓度为(10~(-9)、10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L)。观察给药后肌条张力变化情况，待变化稳定后可以增加一个浓度，继续加药，每次加药前都要在前一浓度达到最大反应稳定后再加入。每次加药的间隔时间约为肌条平衡后10分钟。然后建立累计给药的浓度-反应量效曲线，计算出加药后反应的最大效应及对应浓度。然后彻底冲洗肌条，待张力稳定后向浴槽中加入非选择性DAR拮抗剂（Flupenthixol dihydrochloride），加样浓度与诱导肌条产生最大效应的激动剂浓度相对应。观察其对DAR的拮抗作用。选择性的D-1样受体激动剂（(±)-SKF-3893），选择性的D2R激动剂（(-)-Quinpirolehydrochloride）的加药方法同前。药物诱发的肌条反应均以肌条收缩或舒张百分比的均数±标准误(x±SE)来表示。
结果：
1非选择性DAR激动剂及拮抗剂对人LES的作用非选择性DAR激动剂DA在（10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L）的浓度可以诱导人LES的套索纤维、钩状纤维两种肌条产生收缩效应，在（10~(-5)、10~(-4)、10~(-3)mol/L）的浓度，在收缩反应的同时还可以诱导人LES的套索纤维、钩状纤维两种肌条产生舒张效应。应用非选择性DAR的阻滞剂（Flupenthixol dihydrochloride）（10~(-4)mol/L）可以完全抑制DA（10~(-4)mol/L）所产生的套索纤维、钩状纤维的收缩、舒张效应。
2选择性的D-1样受体激动剂(±)-SKF-3893对人LES的作用选择性的D-1样受体激动剂(±)-SKF-3893在（10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L）浓度下可以诱导人LES的套索纤维、钩状纤维两种肌条产生浓度依赖性的收缩效应。两肌条间收缩效应比较没有统计学差异（F=0.401，P=0.808）。在10~(-4)mol/L时，两肌条均达到最大收缩百分比。钩状纤维的最大收缩百分比为（15.8±2.2）％，套索纤维的最大收缩百分比为（17.8±2.1）％。两者比较无统计学差异（F=0.015，P=0.903）。
3选择性的D2R激动剂(-)-Quinpirole hydrochloride对人LES的作用
选择性的D2R激动剂(-)-Quinpirole hydrochloride在（10~(-5)、10~(-4)、10~(-3)mol/L）浓度下可以诱导人LES的套索纤维、钩状纤维两种肌条产生浓度依赖性的舒张效应，两肌条间舒张效应比较没有统计学差异（F=1.258，P=0.292）。在10~(-3)mol/L时，两肌条均达到最大舒张百分比。钩状纤维的最大舒张百分比为（12.0±1.6）％，套索纤维的最大舒张百分比为（10.8±2.1）％，两者比较无统计学差异（F=0.131，P=0.721）。
结论：
1非选择性DAR激动剂DA低浓度时可以诱导人LES收缩效应，随着其浓度的升高逐渐出现舒张效应。非选择性DAR的阻滞剂Flupenthixoldihydrochloride可以完全抑制非选择性激动剂所诱导的LES的收缩、舒张效应。表明非选择性激动剂是通过DAR来实现其对LES的调节作用。
2选择性的D-1样受体激动剂(±)-SKF-3893可以诱导人LES的两种肌条产生浓度依赖性的收缩效应。在10-4mol/L时，达到最大收缩百分比。两肌条间收缩效应比较没有统计学差异。提示D-1样受体可能参与了LES调节过程中的收缩反应。
3选择性的D2R激动剂(-)-Quinpirole hydrochloride可以诱导人LES的两种肌条产生浓度依赖性的舒张效应，在10-3mol/L时，达到最大舒张百分比。两肌条间舒张效应比较没有统计学差异。提示D2R可能参与了LES调节过程中的舒张反应。
第三部分多巴胺受体在电刺激诱导人食管下括约肌反应中的作用研究
目的：研究选择性DAR的拮抗剂在电场刺激下对人LES的钩状纤维、套索纤维的作用，探讨DAR在LES神经调节通路中发挥的作用。
方法：选取2011年3至2011年11月在河北医科大学第四医院因高位食管中段癌，行食管大部切除术患者20例，其中男性患者14例，女性患者6例，平均年龄58岁。制备钩状纤维和套索纤维肌条，方法同前。将肌条置于含10ml Krebs液的浴槽中，保持37℃恒温条件。持续通以95%O2和5%CO_2的混合气体。将肌条的下端固定于带铂金电极的L形的固定架上。上端与肌肉张力换能器固定，Medlab信号采集系统记录肌条张力变化情况。确保肌条位于两个呈环形且平行的铂金电极中间，其上下端与电极环间的距离应当大于3mm，并将铂金电极与生理药理多用仪连接，准备实施电场刺激。调节肌条长度至最适初长度，方法同前。EFS刺激参数：单脉冲方波、波宽5ms、电压50V、频率1～512Hz以倍数递增。频率从小到大行EFS，计算刺激后的最大效应(E_(max))。刺激停止后待肌条恢复平衡，分别依次向浴槽加入选择性的D-1样受体拮抗剂SCH23390（10~(-4)mol/L）；选择性的D_2R拮抗剂Spiperone hydrochloride（10~(-3)mol/L）。在加药后20分钟后再分别行EFS，比较加药前后EFS刺激人LES出现的反应有无变化。EFS诱发的肌条反应均以肌条的收缩或舒张百分比的均数±标准误(x±SE)来表示。
结果：
1电场刺激对LES钩状和套索纤维的作用：电场刺激可诱导钩状纤维出现频率依赖性舒张，最大舒张时电刺激频率为64Hz，当每一次刺激所引起的舒张反应结束后，肌条立即出现反跳性收缩，亦呈频率依赖模式。EFS诱导钩状纤维舒张的最大舒张百分比为17.1±2.1％。套索纤维对EFS的反应表现为频率依赖性的收缩反应，最大收缩时电刺激频率为128Hz。EFS诱导套索纤维收缩的最大收缩百分比为13.6±1.8％。
2选择性的D-1样受体拮抗剂SCH23390和选择性的D2R拮抗剂Spiperone hydrochloride在EFS引起的人LES钩状纤维频率依赖性舒张反应中的作用：选择性D-1样受体拮抗剂（10~(-4)mol/L）对电场刺激引起的人LES钩状纤维频率依赖性舒张反应无影响，比较用药前后舒张效应无统计学差异（F=1.142，P=0.342）。选择性的D_2R拮抗剂（10~(-3)mol/L）对频率依赖性的舒张反应也无影响，比较用药前后舒张效应无统计学差异（F=0.359，P=0.837）。
3选择性的D-1样受体拮抗剂SCH23390和选择性的D_2R拮抗剂Spiperone hydrochloride在EFS引起的人LES套索纤维频率依赖性收缩反应中的作用：选择性D-1样受体拮抗剂（10~(-4)mol/L）对电场刺激的引起人LES套索纤维频率依赖性收缩反应无影响，比较用药前后收缩效应无统计学差异（F=0.354，P=0.840）。选择性的D_2R拮抗剂（10~(-3)mol/L）对频率依赖性的收缩反应也无影响，比较用药前后收缩效应无统计学差异（F=0.651，P=0.627）。
结论：
1电场刺激可以诱导钩状纤维出现频率依赖性舒张反应。最大舒张时电刺激频率为64Hz。电场刺激可以诱导套索纤维频率依赖性的收缩反应，最大收缩时电刺激频率为128Hz；
2分别应用选择性D-1样受体拮抗剂SCH23390和选择性D_2R拮抗剂Spiperone hydrochloride后，比较用药前后EFS所诱导人LES钩状纤维频率依赖性舒张反应发现，用药前后舒张反应的变化无统计学差异。提示在EFS所诱导的钩状纤维出现的频率依赖性舒张反应中没有D-1样受体的参与。
3分别应用选择性D-1样受体拮抗剂SCH23390和选择性D2R拮抗剂Spiperone hydrochloride后，比较用药前后EFS所诱导人LES套索纤维频率依赖性收缩反应发现，用药前后收缩反应的变化无统计学差异。提示在EFS所诱导的套索纤维出现的频率依赖性收缩反应中没有D2R的参与。
The lower esophageal sphincter (LES) is a thickened region of thecircular muscle layer located at the gastroesophageal junction in human,extending over an axial distance of2–3cm. In1979, Liebermann-Mefferproposed that musculature of equivalent of the LES consists of clasp fibers atthe lesser curvature and sling fibers at the greater curvature. These two musclefibers and the crural diaphragm are in a state of constant or tonic contraction,which is due to both myogenic and neurogenic elements of the LES, and forma high-pressure zone at the gastroesophageal junction to prevent reflux of thegastric contents into the esophagus; on the other hand, swallow as well asesophageal distension induced the transient LES relaxation (TLESR), whichpermits the passage of food from the esophagus into the stomach or to vomitand hiccup, by which induced the reflux of the gastric contents into theesophagus.
The functional regulation of contraction and relaxation of the LES iscompleted by several hormone, neurotransmitter and spontaneous myogenicfactors under the control of the the central nervous system. The vagal efferentfibers synapse with a huge number of enteric motor neurons (EMN), which arelocated in the myenteric plexus (MP), and the can form both inhibitory andexcitatory pathways by the synapse. The preganglionic neurons that form theinhibitory and the excitatory pathway are cholinergic. Furthermore, theneurotransmission of the excitatory postganglionic neurons is cholinergic,whereas the inhibitory postganglionic neurons are nonadrenergicnoncholinergic (NANC). Both the inhibitory and the excitatory vagalpathways exert tonic effects on the LES. The activation of the vagal excitatorypathway can generate contraction of the LES, whereas the activation of thevagal inhibitory pathway can generate relaxation of the LES.
Dopamine (DA), as a predominant catecholamine neurotransmittercontrols, a variety of functions, including locomotor activity, cognition,emotion, endocrine regulation and gastrointestinal motility. All of thesefunctions are mediated by DA receptors, which are important members of theseven transmembrane domain G protein-coupled receptor family. DAR hasbroad expression patterns in both central nervous system and peripherynervous system. By now, five distinct DA receptor subtypes (D1R-D5R) havebeen found. On the basis of their structural, pharmacological, and biochemicalproperties, these receptors were classified as either D1-like dopaminereceptors (D1R and D5R) or D2-class dopamine receptors (D2R, D3R, andD4R). Recent experiments have suggested that DA receptors are widelyexpressed in animal gastrointestinal tract.
Various esophageal motility disorders, such as achalasia, diffuseesophageal spasm and nutcracker esophagus, are all associated with motordisorders of the LES. Recent studies have demonstrated that the regulatorymechanism of the LES involves various receptors, neurotransmitter, and signaltransduction pathways. CCK receptors, and Muscarinic receptors have beendemonstrated to play a role in the regulation of the LES.
Reverse transcription-polymerase chain reaction (RT-PCR), westernblotting, measurement of muscle tension in vitro, and electrical fieldstimulation (EFS) were used to identify expression and function of the DAreceptors in the human LES. The present study investigated the role that theDA receptors play in modulating human LES function. So that we candemonstrate the regulatory mechanism of the LES much more properly, andprovide theoretical bases for the clinical treatment of esophageal motilitydisorders.
PartⅠ Expression of dopamine receptors in human lower esophagealsphincter
Objective: Dopamine (DA) receptors as a member of the Gprotein-coupled receptor family, mediate all of the physiological functions of the catecholaminergic neurotransmitter dopamine. In the present study, weidentified the expression of mRNA and protein of DA receptors in four musclestrips including sling fibers, clasp fibers, circular muscle strips of esophagusand gastric with the use of reverse transcription-polymerase chain reaction(RT-PCR) and western blot, to investigated the role that the DA receptors playin modulating human LES function.
Methods: Thirty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between December2007and October2008were selected in thisstudy. There were23male and7female patients, with a mean age of64years.Each specimen including part of the gastric fundus, the gastroesophagealjunction, and the esophageal body, was resected en bloc in the operating room.After the mucosa and submucosa were removed by sharp dissection, the slingand clasp fibers of LES, and circular muscle strips of esophagus and gastricwere obtained from various regions of the gastroesophageal junction andadjacent structures. The dissected muscle strips were frozen in liquid nitrogenand stored at-80°C for subsequent RNA and protein extraction. Total RNAwas extracted by acid guanidinium thiocyanate-phenolchloroform extraction.After the identification of its purity and integrity with the use of ultravioletspectrophotometer and1%agarose gels, reverse transcription-polymerasechain reaction (RT-PCR) was performed using primers designed specificallyto match the DA receptors’ mRNA, to investigate the mRNA expression ofDA receptors. The unit of integrated optical density (IOD) of the amplifiedproducts was calculated with Gel-Pro software. The expression of DAreceptor mRNA was expressed by the ratio of IOD value of DA receptor bandto β-actin band. The integral membrane protein receptors, which wereextracted from muscle tissue, were quantitated and adjusted to the identicalconcentration, the different DA receptors were separated by electrophoresis.At last the detection of the protein expression was operated using different DAreceptors’ polyclonal antibody after the trarsmembrane. The IOD value wasalso calculated with the Gel-Pro software.
Results: The value of A260/280of total RNA was between1.6and1.8after ultraviolet spectrophotometry. The width and brightness of28S bandwere double than18S band in1%agarose gels. The band of β-actin mRNAwas uniformly838bp. Transcripts for D1R, D2R, and D5R were identified inthe four muscle strips. D3R and D4R mRNA were not identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent DA receptors’ mRNA in the same muscle strips (F=669.00, P=0.00). The rank order of the extent of expression was D_1R>D_5R> D_2R.However, there was no significant difference in mRNA expression of DAreceptors between the four muscle strips.(F=0.18, P=0.90). Proteinexpression of three DA receptor subtypes were identified, they were D_1R, D_2Rand D_5R, with respective molecular,51KD,51KD and53KD. D3R and D4Rprotein expression were not identified in the four muscle strips. There was asignificant difference in IOD values for different DA receptors in the samemuscle strip (F=84.53, P=0.00). The rank order of the value was the sameas the result of the RT-PCR. There was no significant difference in IOD valuesbetween the four muscle strips.(F=0.10, P=0.96).
Conclusion: D_1R，D_2R，D5R can be detected in the human LES, the rankorder of the extent of expression is D1R>D5R> D2R, and probably contributeto LES function. D_3R and D_4R are not expressed, and probably do notcontribute to LES function in humans.
PartⅡ The role of dopamine receptors in modulating human loweresophageal sphincter
Objective: To identify the effect that non-selective dopamine receptoragonist, antagonist and selective dopamine receptor agonist have played inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES), and investigate the role of dopamine receptor subtypes inmodulating contraction and relaxation of the LES.
Methods: Thirty patients who underwent esophago-gastrectomy for mid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between August2010and March2011were selected in this study.There were17male and13female patients, with a mean age of62years. Eachspecimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. In the laboratory, the surgical specimen wasopened along the long axis of the esophagus and the greater curvature of thestomach. It was washed with37°C Krebs solution which had previously beenbubbled with95%O2and50%CO2. The specimen was then pinned on a waxplate with the mucosal surface facing up to maintain its approximate in situdimensions. Then both the mucosa and submucosa were removed by sharpdissection. The LES was recognized as a thickened band of circular muscle atthe gastroesophageal junction. The sling and clasp fibers could be identified asthickened bands of circular oriented smooth muscle in the gastric cardia,adjacent to the greater and lesser curvature of the stomach, respectively.Transversely oriented strips measuring2mm×10mm were prepared from thesling and clasp fibers so that the long axis of the muscle strip paralleled thelong axis of smooth muscle cells constituting the circular muscle layer. Eachend of the muscle strips were attached with silk suture, and one of the end wastied to to an isometric force transducer supported on a rack-and-pinion clampto facilitate an accurate length adjustment of the muscle strips. Tension wasrecorded the software of MedLab6.0. The strips were incubated1h in the10-ml jacketed tissue baths filled with Krebs solution maintained at37°C andbubbled continuously with95%O2-5%CO2. Each muscle strip was stretchedslightly and rapidly until200mg of force was generated. This was taken asthe initial length (L0). The muscle strips were then sequentially stretched to200%of the L0, at the increments of25%of the L0each time. This was takenas the most suitable initial length. The muscle strips were equilibrated for40min in in the10-ml jacketed tissue baths filled with Krebs solution maintainedat37°C and bubbled continuously with95%O2-5%CO2. The administration of non-selective dopamine receptor agonist Dopamine hydrochloride (DA)was a cumulative manner from10-9to10-3mol/L, each administration beingdone when the response of the previous concentration reached a maximum.The concentration of the non-selective dopamine receptor antagonistFlupenthixol dihydrochloride was identical with optimal concentration leadingto maximum response. All of the drug concentrations are final concentrationsin the tissue bath. The administration of selective D-1like receptor agonist(±)-SKF-3893and selective D2R agonist (-)-Quinpirole hydrochloride were inthe same way. The responses in all of the experiments were quantified basedupon a percentage of the baseline value of muscle strip tone relative to thenadir of the response. The data were expressed as means±standard error.
Results:
1Effect of non-selective dopamine receptor agonist and antagonist on thehuman LES
The non-selective dopamine receptor agonist DA induced the contractionof the clasp and sling fibers of the human LES at the concentration of (10-7,10~(-6),10~(-5),10~(-4),10~(-3)mol/L), while relaxation induced at the concentration of(10~(-5),10~(-4),10~(-3)mol/L). The response induced by non-selective dopaminereceptor agonist Flupenthixol dihydrochloride at the concentration of10-4mol/L, was inhibited completely by non-selective dopamine receptorantagonist Flupenthixol dihydrochloride (10~(-4)mol/L).
2Effect of selective D-1like receptor agonist on the human LES
The selective D-1like receptor agonist (±)-SKF-3893induced aconcentration-dependent contractile response of the clasp and sling fibers ofthe human LES at the concentration of (10~(-7),10~(-6),10~(-5),10~(-4),10~(-3)mol/L). Therewas no significant difference in contraction between the sling fibers and claspfibers (F=0.401, P=0.808). The optimal concentration leading to maximumcontraction was10~(-4)mol/L. The maximum contraction of clasp fibers was(15.8±2.2)％. The maximum contraction of sling fibers was (17.8±2.1)％.There was no significant difference (F=0.015, P=0.903).
3Effect of selective D2R agonist on the human LES
The selective D_2R agonist (-)-Quinpirole hydrochloride inducedrelaxation of the human LES at the concentration of (10-5,10-4,10-3mol/L),which was also in a concentration-dependent manner. There was no significantdifference in relaxation between the sling fibers and clasp fibers (F=1.258,P=0.292). The optimal concentration leading to maximum relaxation was10~(-3)mol/l. The maximum relaxation of clasp fibers was (12.0±1.6)％. Themaximum relaxation of sling fibers was (10.8±2.1)％. There was nosignificant difference (F=0.131, P=0.721).
Conclusion:
1The non-selective dopamine receptor agonist can induce the contraction ofthe human LES at a low concentration, relaxation occurs with the rise of theconcentration. The reaction of the human LES can be inhibited completely bynon-selective dopamine receptor antagonist. This study indicates that DARinvolves the reaction of LES to DA.
2The selective D-1-like receptor agonist induces a concentration-dependentcontractile response. The optimal concentration leading to maximumcontraction is10-4mol/l. There is no significant difference in contractionbetween the sling fibers and clasp fibers. This study indicates that the D-1-likereceptor is involved in the contractile response of the human LES.
3The selective D_2R agonist induces relaxation of the human LES, which isalso in a concentration-dependent manner. The optimal concentration leadingto maximum relaxation is10~(-3)mol/l. There is no significant difference inrelaxation between the sling fibers and clasp fibers. This study indicates thatD2R is involved in the relaxation of the human LES.
PartⅢ The contribution of dopamine receptors in the response ofhuman lower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that dopamine receptor subtypes play inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES) under the electical field stimulation (EFS), and investigate therole of dopamine receptor subtypes in vagal pathways that modulating human LES function.
Methods: Twenty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between March2011and November2011were selected in thisstudy. There were14male and6female patients, with a mean age of58years.Each specimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. The sling and clasp muscle strips wereprepared using similar methods to those we have described previously. Eachend of the muscle strips were attached with silk suture. The muscle strip wastied at one end to the electrode holder and at the other end was tied to to anisometric force transducer supported on a rack-and-pinion clamp to facilitatean accurate length adjustment of the muscle strips. Tension was recorded thesoftware of MedLab6.0. The muscle strips were mounted through concentricplatinum electrodes, makesure the distance between the end and electrode wasmore than3mm. The electrodes were connected to the output of thestimulator that delivered single pulse square wave, trains of0.5ms,50-Vsquare wave pulses at1～512Hz. Before the study, each muscle strip wasgently stretched to the the most suitable initial length, just like what we haveshown previously. The responses of muscle strips to EFS were sequentiallyassessed over a frequency range of1～512Hz, with5min between eachfrequency under control conditions. Then the muscle strip was stimulatedagain after20min of administration of selective D-1like receptor antagonistSCH23390at the concentration of10-4mol/L. The administration of selectiveD2R antagonist Spiperone hydrochloride at the concentration of10-3mol/Lwas in the same way. The responses in all of the experiments were quantifiedbased upon a percentage of the baseline value of muscle strip tone relative tothe nadir of the response. The data were expressed as means±standard error.
Results:
1Effect of the EFS on the the clasp and sling fibers of the human LES
The EFS induced a frequency-dependent relaxation in clasp fibers. Aftereach of the stimulation, there was a quick rebound contraction which was alsoin the frequency-dependent manner. The optimal frequency resulting inmaximum relaxation was64Hz. The EFS induced a frequency-dependentcontraction in sling fibers. The optimal frequency resulting in maximumcontraction was128Hz. The maximum contraction was (13.6±1.8)％.
2Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the clasp fibers of thehuman LES under the EFS
The selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent relaxation in claspfibers of the human LES induced by the EFS (F=1.142, P=0.342). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS (F=0.359, P=0.837).
3Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the sling fibers of thehuman LES under the EFS
The selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent contraction insling fibers of the human LES induced by the EFS (F=0.354, P=0.840). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent contraction in sling fibers of the human LES induced bythe EFS (F=0.651, P=0.627).
Conclusion:
1The EFS induces frequency-dependent relaxation in clasp fibers. Theoptimal frequency resulting in maximum relaxation is64Hz. EFS inducesfrequency-dependent contraction in the sling fibers. The optimal frequencyleading to maximum contraction is128Hz.
2Both of the selective D-1like dopamine receptor antagonist and theselective D_2R antagonist produces no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of clasp fiers of the human LES induced by the EFS.
3Both of the selective D-1like dopamine receptor antagonist and theselective D2R antagonist produces no significant change in thefrequency-dependent contraction in the sling fibers of the human LES inducedby the EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of sling fiers of the human LES induced by the EFS.
LES的收缩和舒张的功能调节机制是在中枢神经系统的支配下，由多种激素、神经递质及自身肌源性因素共同参与完成的。一般认为迷走神经输出神经末梢与LES肌层肠道运动神经元（Enteric Motor Neurons, EMN）形成突触联系，共同构成了调节LES收缩和舒张的兴奋性迷走神经通路和抑制性迷走神经通路。兴奋性迷走神经通路由节前胆碱能神经元和节后胆碱能神经元共同构成。该通路的激活可以引起LES收缩。而抑制性迷走神经通路则是由节前胆碱能神经元和节后非肾上腺非胆碱能（NonadrenergicNoncholinergic, NANC）神经元构成。激活抑制性通路可以引起LES舒张。
多巴胺（Dopamine, DA）是儿茶酚胺类神经递质之一，在自主运动、认知、情感、激素调节以及胃肠运动等功能的调节中发挥着重要的作用。这些功能的调节均是通过多巴胺受体（Dopamine receptor, DAR）实现的。DAR作为含有七跨膜结构域G蛋白偶联受体家族重要的受体类型之一，广泛存在于哺乳动物的中枢及外周神经系统。到目前为止已经发现DAR共有D1R-D5R五种亚型，按照各自的结构，药理学性质以及生物学属性可以将这些受体亚型分为D1样受体（D1R、D5R）和D2样受体（D2R、D3R、D4R）。近些年的研究表明，DAR广泛地分布于动物的胃肠道之中。
多种食管运动功能异常性疾病如贲门失弛缓、弥漫性食管痉挛、胡桃夹食管等均与LES的运动异常有关，目前研究表明LES的调节机制涉及多种受体，神经递质和信号转导通路。研究表明胆囊收缩素受体（Cholecystokinin receptor），毒蕈碱受体（Muscarinic receptor）均在LES的调节机制中发挥着重要的作用。
本研究采用逆转录聚合酶链反应(RT-PCR)，蛋白印迹法（Western-blot），离体肌张力测定技术以及电场刺激（Electrical fieldstimulation, EFS）等方法对DAR在人LES中的表达及功能进行研究。探讨了DAR在人LES调节机制中的作用，为进一步研究DA及其受体在人LES调节机制中的作用奠定基础，从而更为完善的阐述人LES调节机制，为食管运动功能障碍性疾病的临床治疗提供理论依据。
第一部分多巴胺受体在人食管下括约肌的表达研究目的：DAR是G蛋白偶联受体家族重要的受体类型之一，介导着儿茶酚胺类神经递质DA所有的生理学功能。本试验采用逆转录聚合酶链反应(RT-PCR)和蛋白印迹法（Western-blot）研究人LES的DAR的5种亚型在钩状纤维、套索纤维、食管和胃底的环行肌中mRNA、受体蛋白的表达规律，探讨DAR在人LES的功能调节中发挥的作用。
方法：选取2007年12月至2008年10月在河北医科大学第四医院因高位食管中段癌，而行食管大部切除术的患者共30例。其中23名男性，7名女性，平均年龄64岁。手术室收集新鲜EGJ手术标本，实验室锐性剥去食管下段、贲门及胃底的粘膜及粘膜下层后，制备钩状纤维、套索纤维、食管和胃底环行肌的肌条。提取组织RNA，经紫外分光光度计和1％琼脂糖凝胶电泳鉴定其纯度和完整性之后，分别应用5种DAR亚型的引物行RT-PCR，检测其mRNA在4种肌条的表达，用Gel Pro软件分析扩增产物的光密度值(IOD)，并计算DAR亚型与β-actin IOD的比值表示肌条中mRNA的相对含量。提取组织蛋白，经考马斯亮蓝G-250定量，将蛋白调整至相同浓度。然后应用电泳仪电泳，分离出与DAR各个亚型相应的受体蛋白后转膜，转膜结束后分别应用DAR各个亚型的抗体进行孵育及DAB染色，经Gel Pro软件分析DAB反应条带的光密度值(IOD)。
结果：紫外分光度计测定显示，总RNAA260/A280比值为1.6-1.8；1%琼脂糖凝胶电泳可见28S条带的宽度和亮度均是18S条带的两倍。内参β-actin于838bp处亮度均匀，在各个反应中，内参β-actin的PCR扩增结果一致。3种DAR亚型的mRNA在四种肌条中均可见表达，分别是D_1R，D_2R，D_5R。D_3R，D_4R的mRNA在四种肌条内均未见表达。PCR扩增产物的长度与其设计长度完全一致。同一肌条不同DAR亚型的mRNA表达水平的比较有统计学差异(F=669.00, P=0.00)，其表达水平的强度依次为：D1R＞D5R＞D2R。同一DAR亚型的mRNA表达水平在不同的肌条间的比较没有统计学差异(F=0.18, P=0.90)。有3种DAR的受体亚型的蛋白在四种肌条内均可见表达，分别是D1R，D2R，D5R，其分子量大小依次为51KD、51KD、53KD。D3R，D4R的蛋白在四种肌条内均未见表达。同一肌条不同DAR亚型的蛋白表达水平的比较有统计学差异(F=84.53, P=0.00)，其蛋白表达水平的强弱与mRNA表达水平的结果一致。同一DAR亚型的蛋白表达水平在不同的肌条间比较没有统计学差异(F=0.10, P=0.96)。
结论：人LES存在3种DAR亚型，分别是D1R，D2R，D5R。其表达水平的高低依次为D1R>D5R> D2R，可能在LES的功能调节中发挥着作用。D3R和D4R在人LES中无表达，可能对LES的功能调节没有影响。
第二部分多巴胺受体在人食管下括约肌调节机制中的功能研究
目的：研究非选择性DAR的激动剂和拮抗剂以及选择性的DAR的激动剂对人离体LES的钩状纤维（clasp）、套索纤维（sling）的作用，探讨DAR在人LES收缩和舒张的调节机制中所发挥的的作用。
方法：选取2010年8月到2011年3月在河北医科大学第四医院因高位食管中段癌行食管大部切除术患者30例。男性患者17例，女性患者13例，平均年龄62岁。手术室采集新鲜标本，于标记处沿胃大弯侧切开标本，粘膜面向上固定于盛有Krebs液的蜡盘中，持续通以95％O2和5％CO_2的混合气体。锐性剥离胃贲门部以及食管下段的粘膜层及粘膜下层，可见食管胃结合部的发白增厚的肌肉环，即为LES。肉眼识别出胃小弯侧位于标本的中央呈半环形的钩状纤维（clasp）与胃大弯侧位于标本的两端并呈斜行的套索纤维（sling）。沿着各自肌纤维走行的方向锐性游离钩状纤维（clasp）和套索纤维（sling），制备2mm×10mm的肌条。应用丝线分别将肌条的两端扎紧，置于含Krebs液10ml的浴槽中，保持37℃恒温条件。持续通以95%O2和5%CO2的混合气体。肌条下端固定，上端固定于JZ101型肌肉张力换能器，经Medlab信号采集系统记录张力变化情况。轻微缓慢牵拉肌条使之张力维持于200mg，此时的肌条长度作为初始长度L0，然后多次缓慢牵拉肌条，每次增加其初始长度L0的25％左右，每次待被动张力稳定15min后，再次牵拉。如此反复，每次牵拉间隔约20min，直至牵拉肌条长度至初始长度L0的200％为最适初长度。待肌条最适初长度稳定约40分钟后，以浓度累积方式向浴槽中加入非选择性DAR激动剂（Dopamine hydrochloride, DA），给药浓度为(10~(-9)、10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L)。观察给药后肌条张力变化情况，待变化稳定后可以增加一个浓度，继续加药，每次加药前都要在前一浓度达到最大反应稳定后再加入。每次加药的间隔时间约为肌条平衡后10分钟。然后建立累计给药的浓度-反应量效曲线，计算出加药后反应的最大效应及对应浓度。然后彻底冲洗肌条，待张力稳定后向浴槽中加入非选择性DAR拮抗剂（Flupenthixol dihydrochloride），加样浓度与诱导肌条产生最大效应的激动剂浓度相对应。观察其对DAR的拮抗作用。选择性的D-1样受体激动剂（(±)-SKF-3893），选择性的D2R激动剂（(-)-Quinpirolehydrochloride）的加药方法同前。药物诱发的肌条反应均以肌条收缩或舒张百分比的均数±标准误(x±SE)来表示。
结果：
1非选择性DAR激动剂及拮抗剂对人LES的作用非选择性DAR激动剂DA在（10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L）的浓度可以诱导人LES的套索纤维、钩状纤维两种肌条产生收缩效应，在（10~(-5)、10~(-4)、10~(-3)mol/L）的浓度，在收缩反应的同时还可以诱导人LES的套索纤维、钩状纤维两种肌条产生舒张效应。应用非选择性DAR的阻滞剂（Flupenthixol dihydrochloride）（10~(-4)mol/L）可以完全抑制DA（10~(-4)mol/L）所产生的套索纤维、钩状纤维的收缩、舒张效应。
2选择性的D-1样受体激动剂(±)-SKF-3893对人LES的作用选择性的D-1样受体激动剂(±)-SKF-3893在（10~(-7)、10~(-6)、10~(-5)、10~(-4)、10~(-3)mol/L）浓度下可以诱导人LES的套索纤维、钩状纤维两种肌条产生浓度依赖性的收缩效应。两肌条间收缩效应比较没有统计学差异（F=0.401，P=0.808）。在10~(-4)mol/L时，两肌条均达到最大收缩百分比。钩状纤维的最大收缩百分比为（15.8±2.2）％，套索纤维的最大收缩百分比为（17.8±2.1）％。两者比较无统计学差异（F=0.015，P=0.903）。
3选择性的D2R激动剂(-)-Quinpirole hydrochloride对人LES的作用
选择性的D2R激动剂(-)-Quinpirole hydrochloride在（10~(-5)、10~(-4)、10~(-3)mol/L）浓度下可以诱导人LES的套索纤维、钩状纤维两种肌条产生浓度依赖性的舒张效应，两肌条间舒张效应比较没有统计学差异（F=1.258，P=0.292）。在10~(-3)mol/L时，两肌条均达到最大舒张百分比。钩状纤维的最大舒张百分比为（12.0±1.6）％，套索纤维的最大舒张百分比为（10.8±2.1）％，两者比较无统计学差异（F=0.131，P=0.721）。
结论：
1非选择性DAR激动剂DA低浓度时可以诱导人LES收缩效应，随着其浓度的升高逐渐出现舒张效应。非选择性DAR的阻滞剂Flupenthixoldihydrochloride可以完全抑制非选择性激动剂所诱导的LES的收缩、舒张效应。表明非选择性激动剂是通过DAR来实现其对LES的调节作用。
2选择性的D-1样受体激动剂(±)-SKF-3893可以诱导人LES的两种肌条产生浓度依赖性的收缩效应。在10-4mol/L时，达到最大收缩百分比。两肌条间收缩效应比较没有统计学差异。提示D-1样受体可能参与了LES调节过程中的收缩反应。
3选择性的D2R激动剂(-)-Quinpirole hydrochloride可以诱导人LES的两种肌条产生浓度依赖性的舒张效应，在10-3mol/L时，达到最大舒张百分比。两肌条间舒张效应比较没有统计学差异。提示D2R可能参与了LES调节过程中的舒张反应。
第三部分多巴胺受体在电刺激诱导人食管下括约肌反应中的作用研究
目的：研究选择性DAR的拮抗剂在电场刺激下对人LES的钩状纤维、套索纤维的作用，探讨DAR在LES神经调节通路中发挥的作用。
方法：选取2011年3至2011年11月在河北医科大学第四医院因高位食管中段癌，行食管大部切除术患者20例，其中男性患者14例，女性患者6例，平均年龄58岁。制备钩状纤维和套索纤维肌条，方法同前。将肌条置于含10ml Krebs液的浴槽中，保持37℃恒温条件。持续通以95%O2和5%CO_2的混合气体。将肌条的下端固定于带铂金电极的L形的固定架上。上端与肌肉张力换能器固定，Medlab信号采集系统记录肌条张力变化情况。确保肌条位于两个呈环形且平行的铂金电极中间，其上下端与电极环间的距离应当大于3mm，并将铂金电极与生理药理多用仪连接，准备实施电场刺激。调节肌条长度至最适初长度，方法同前。EFS刺激参数：单脉冲方波、波宽5ms、电压50V、频率1～512Hz以倍数递增。频率从小到大行EFS，计算刺激后的最大效应(E_(max))。刺激停止后待肌条恢复平衡，分别依次向浴槽加入选择性的D-1样受体拮抗剂SCH23390（10~(-4)mol/L）；选择性的D_2R拮抗剂Spiperone hydrochloride（10~(-3)mol/L）。在加药后20分钟后再分别行EFS，比较加药前后EFS刺激人LES出现的反应有无变化。EFS诱发的肌条反应均以肌条的收缩或舒张百分比的均数±标准误(x±SE)来表示。
结果：
1电场刺激对LES钩状和套索纤维的作用：电场刺激可诱导钩状纤维出现频率依赖性舒张，最大舒张时电刺激频率为64Hz，当每一次刺激所引起的舒张反应结束后，肌条立即出现反跳性收缩，亦呈频率依赖模式。EFS诱导钩状纤维舒张的最大舒张百分比为17.1±2.1％。套索纤维对EFS的反应表现为频率依赖性的收缩反应，最大收缩时电刺激频率为128Hz。EFS诱导套索纤维收缩的最大收缩百分比为13.6±1.8％。
2选择性的D-1样受体拮抗剂SCH23390和选择性的D2R拮抗剂Spiperone hydrochloride在EFS引起的人LES钩状纤维频率依赖性舒张反应中的作用：选择性D-1样受体拮抗剂（10~(-4)mol/L）对电场刺激引起的人LES钩状纤维频率依赖性舒张反应无影响，比较用药前后舒张效应无统计学差异（F=1.142，P=0.342）。选择性的D_2R拮抗剂（10~(-3)mol/L）对频率依赖性的舒张反应也无影响，比较用药前后舒张效应无统计学差异（F=0.359，P=0.837）。
3选择性的D-1样受体拮抗剂SCH23390和选择性的D_2R拮抗剂Spiperone hydrochloride在EFS引起的人LES套索纤维频率依赖性收缩反应中的作用：选择性D-1样受体拮抗剂（10~(-4)mol/L）对电场刺激的引起人LES套索纤维频率依赖性收缩反应无影响，比较用药前后收缩效应无统计学差异（F=0.354，P=0.840）。选择性的D_2R拮抗剂（10~(-3)mol/L）对频率依赖性的收缩反应也无影响，比较用药前后收缩效应无统计学差异（F=0.651，P=0.627）。
结论：
1电场刺激可以诱导钩状纤维出现频率依赖性舒张反应。最大舒张时电刺激频率为64Hz。电场刺激可以诱导套索纤维频率依赖性的收缩反应，最大收缩时电刺激频率为128Hz；
2分别应用选择性D-1样受体拮抗剂SCH23390和选择性D_2R拮抗剂Spiperone hydrochloride后，比较用药前后EFS所诱导人LES钩状纤维频率依赖性舒张反应发现，用药前后舒张反应的变化无统计学差异。提示在EFS所诱导的钩状纤维出现的频率依赖性舒张反应中没有D-1样受体的参与。
3分别应用选择性D-1样受体拮抗剂SCH23390和选择性D2R拮抗剂Spiperone hydrochloride后，比较用药前后EFS所诱导人LES套索纤维频率依赖性收缩反应发现，用药前后收缩反应的变化无统计学差异。提示在EFS所诱导的套索纤维出现的频率依赖性收缩反应中没有D2R的参与。
The lower esophageal sphincter (LES) is a thickened region of thecircular muscle layer located at the gastroesophageal junction in human,extending over an axial distance of2–3cm. In1979, Liebermann-Mefferproposed that musculature of equivalent of the LES consists of clasp fibers atthe lesser curvature and sling fibers at the greater curvature. These two musclefibers and the crural diaphragm are in a state of constant or tonic contraction,which is due to both myogenic and neurogenic elements of the LES, and forma high-pressure zone at the gastroesophageal junction to prevent reflux of thegastric contents into the esophagus; on the other hand, swallow as well asesophageal distension induced the transient LES relaxation (TLESR), whichpermits the passage of food from the esophagus into the stomach or to vomitand hiccup, by which induced the reflux of the gastric contents into theesophagus.
The functional regulation of contraction and relaxation of the LES iscompleted by several hormone, neurotransmitter and spontaneous myogenicfactors under the control of the the central nervous system. The vagal efferentfibers synapse with a huge number of enteric motor neurons (EMN), which arelocated in the myenteric plexus (MP), and the can form both inhibitory andexcitatory pathways by the synapse. The preganglionic neurons that form theinhibitory and the excitatory pathway are cholinergic. Furthermore, theneurotransmission of the excitatory postganglionic neurons is cholinergic,whereas the inhibitory postganglionic neurons are nonadrenergicnoncholinergic (NANC). Both the inhibitory and the excitatory vagalpathways exert tonic effects on the LES. The activation of the vagal excitatorypathway can generate contraction of the LES, whereas the activation of thevagal inhibitory pathway can generate relaxation of the LES.
Dopamine (DA), as a predominant catecholamine neurotransmittercontrols, a variety of functions, including locomotor activity, cognition,emotion, endocrine regulation and gastrointestinal motility. All of thesefunctions are mediated by DA receptors, which are important members of theseven transmembrane domain G protein-coupled receptor family. DAR hasbroad expression patterns in both central nervous system and peripherynervous system. By now, five distinct DA receptor subtypes (D1R-D5R) havebeen found. On the basis of their structural, pharmacological, and biochemicalproperties, these receptors were classified as either D1-like dopaminereceptors (D1R and D5R) or D2-class dopamine receptors (D2R, D3R, andD4R). Recent experiments have suggested that DA receptors are widelyexpressed in animal gastrointestinal tract.
Various esophageal motility disorders, such as achalasia, diffuseesophageal spasm and nutcracker esophagus, are all associated with motordisorders of the LES. Recent studies have demonstrated that the regulatorymechanism of the LES involves various receptors, neurotransmitter, and signaltransduction pathways. CCK receptors, and Muscarinic receptors have beendemonstrated to play a role in the regulation of the LES.
Reverse transcription-polymerase chain reaction (RT-PCR), westernblotting, measurement of muscle tension in vitro, and electrical fieldstimulation (EFS) were used to identify expression and function of the DAreceptors in the human LES. The present study investigated the role that theDA receptors play in modulating human LES function. So that we candemonstrate the regulatory mechanism of the LES much more properly, andprovide theoretical bases for the clinical treatment of esophageal motilitydisorders.
PartⅠ Expression of dopamine receptors in human lower esophagealsphincter
Objective: Dopamine (DA) receptors as a member of the Gprotein-coupled receptor family, mediate all of the physiological functions of the catecholaminergic neurotransmitter dopamine. In the present study, weidentified the expression of mRNA and protein of DA receptors in four musclestrips including sling fibers, clasp fibers, circular muscle strips of esophagusand gastric with the use of reverse transcription-polymerase chain reaction(RT-PCR) and western blot, to investigated the role that the DA receptors playin modulating human LES function.
Methods: Thirty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between December2007and October2008were selected in thisstudy. There were23male and7female patients, with a mean age of64years.Each specimen including part of the gastric fundus, the gastroesophagealjunction, and the esophageal body, was resected en bloc in the operating room.After the mucosa and submucosa were removed by sharp dissection, the slingand clasp fibers of LES, and circular muscle strips of esophagus and gastricwere obtained from various regions of the gastroesophageal junction andadjacent structures. The dissected muscle strips were frozen in liquid nitrogenand stored at-80°C for subsequent RNA and protein extraction. Total RNAwas extracted by acid guanidinium thiocyanate-phenolchloroform extraction.After the identification of its purity and integrity with the use of ultravioletspectrophotometer and1%agarose gels, reverse transcription-polymerasechain reaction (RT-PCR) was performed using primers designed specificallyto match the DA receptors’ mRNA, to investigate the mRNA expression ofDA receptors. The unit of integrated optical density (IOD) of the amplifiedproducts was calculated with Gel-Pro software. The expression of DAreceptor mRNA was expressed by the ratio of IOD value of DA receptor bandto β-actin band. The integral membrane protein receptors, which wereextracted from muscle tissue, were quantitated and adjusted to the identicalconcentration, the different DA receptors were separated by electrophoresis.At last the detection of the protein expression was operated using different DAreceptors’ polyclonal antibody after the trarsmembrane. The IOD value wasalso calculated with the Gel-Pro software.
Results: The value of A260/280of total RNA was between1.6and1.8after ultraviolet spectrophotometry. The width and brightness of28S bandwere double than18S band in1%agarose gels. The band of β-actin mRNAwas uniformly838bp. Transcripts for D1R, D2R, and D5R were identified inthe four muscle strips. D3R and D4R mRNA were not identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent DA receptors’ mRNA in the same muscle strips (F=669.00, P=0.00). The rank order of the extent of expression was D_1R>D_5R> D_2R.However, there was no significant difference in mRNA expression of DAreceptors between the four muscle strips.(F=0.18, P=0.90). Proteinexpression of three DA receptor subtypes were identified, they were D_1R, D_2Rand D_5R, with respective molecular,51KD,51KD and53KD. D3R and D4Rprotein expression were not identified in the four muscle strips. There was asignificant difference in IOD values for different DA receptors in the samemuscle strip (F=84.53, P=0.00). The rank order of the value was the sameas the result of the RT-PCR. There was no significant difference in IOD valuesbetween the four muscle strips.(F=0.10, P=0.96).
Conclusion: D_1R，D_2R，D5R can be detected in the human LES, the rankorder of the extent of expression is D1R>D5R> D2R, and probably contributeto LES function. D_3R and D_4R are not expressed, and probably do notcontribute to LES function in humans.
PartⅡ The role of dopamine receptors in modulating human loweresophageal sphincter
Objective: To identify the effect that non-selective dopamine receptoragonist, antagonist and selective dopamine receptor agonist have played inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES), and investigate the role of dopamine receptor subtypes inmodulating contraction and relaxation of the LES.
Methods: Thirty patients who underwent esophago-gastrectomy for mid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between August2010and March2011were selected in this study.There were17male and13female patients, with a mean age of62years. Eachspecimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. In the laboratory, the surgical specimen wasopened along the long axis of the esophagus and the greater curvature of thestomach. It was washed with37°C Krebs solution which had previously beenbubbled with95%O2and50%CO2. The specimen was then pinned on a waxplate with the mucosal surface facing up to maintain its approximate in situdimensions. Then both the mucosa and submucosa were removed by sharpdissection. The LES was recognized as a thickened band of circular muscle atthe gastroesophageal junction. The sling and clasp fibers could be identified asthickened bands of circular oriented smooth muscle in the gastric cardia,adjacent to the greater and lesser curvature of the stomach, respectively.Transversely oriented strips measuring2mm×10mm were prepared from thesling and clasp fibers so that the long axis of the muscle strip paralleled thelong axis of smooth muscle cells constituting the circular muscle layer. Eachend of the muscle strips were attached with silk suture, and one of the end wastied to to an isometric force transducer supported on a rack-and-pinion clampto facilitate an accurate length adjustment of the muscle strips. Tension wasrecorded the software of MedLab6.0. The strips were incubated1h in the10-ml jacketed tissue baths filled with Krebs solution maintained at37°C andbubbled continuously with95%O2-5%CO2. Each muscle strip was stretchedslightly and rapidly until200mg of force was generated. This was taken asthe initial length (L0). The muscle strips were then sequentially stretched to200%of the L0, at the increments of25%of the L0each time. This was takenas the most suitable initial length. The muscle strips were equilibrated for40min in in the10-ml jacketed tissue baths filled with Krebs solution maintainedat37°C and bubbled continuously with95%O2-5%CO2. The administration of non-selective dopamine receptor agonist Dopamine hydrochloride (DA)was a cumulative manner from10-9to10-3mol/L, each administration beingdone when the response of the previous concentration reached a maximum.The concentration of the non-selective dopamine receptor antagonistFlupenthixol dihydrochloride was identical with optimal concentration leadingto maximum response. All of the drug concentrations are final concentrationsin the tissue bath. The administration of selective D-1like receptor agonist(±)-SKF-3893and selective D2R agonist (-)-Quinpirole hydrochloride were inthe same way. The responses in all of the experiments were quantified basedupon a percentage of the baseline value of muscle strip tone relative to thenadir of the response. The data were expressed as means±standard error.
Results:
1Effect of non-selective dopamine receptor agonist and antagonist on thehuman LES
The non-selective dopamine receptor agonist DA induced the contractionof the clasp and sling fibers of the human LES at the concentration of (10-7,10~(-6),10~(-5),10~(-4),10~(-3)mol/L), while relaxation induced at the concentration of(10~(-5),10~(-4),10~(-3)mol/L). The response induced by non-selective dopaminereceptor agonist Flupenthixol dihydrochloride at the concentration of10-4mol/L, was inhibited completely by non-selective dopamine receptorantagonist Flupenthixol dihydrochloride (10~(-4)mol/L).
2Effect of selective D-1like receptor agonist on the human LES
The selective D-1like receptor agonist (±)-SKF-3893induced aconcentration-dependent contractile response of the clasp and sling fibers ofthe human LES at the concentration of (10~(-7),10~(-6),10~(-5),10~(-4),10~(-3)mol/L). Therewas no significant difference in contraction between the sling fibers and claspfibers (F=0.401, P=0.808). The optimal concentration leading to maximumcontraction was10~(-4)mol/L. The maximum contraction of clasp fibers was(15.8±2.2)％. The maximum contraction of sling fibers was (17.8±2.1)％.There was no significant difference (F=0.015, P=0.903).
3Effect of selective D2R agonist on the human LES
The selective D_2R agonist (-)-Quinpirole hydrochloride inducedrelaxation of the human LES at the concentration of (10-5,10-4,10-3mol/L),which was also in a concentration-dependent manner. There was no significantdifference in relaxation between the sling fibers and clasp fibers (F=1.258,P=0.292). The optimal concentration leading to maximum relaxation was10~(-3)mol/l. The maximum relaxation of clasp fibers was (12.0±1.6)％. Themaximum relaxation of sling fibers was (10.8±2.1)％. There was nosignificant difference (F=0.131, P=0.721).
Conclusion:
1The non-selective dopamine receptor agonist can induce the contraction ofthe human LES at a low concentration, relaxation occurs with the rise of theconcentration. The reaction of the human LES can be inhibited completely bynon-selective dopamine receptor antagonist. This study indicates that DARinvolves the reaction of LES to DA.
2The selective D-1-like receptor agonist induces a concentration-dependentcontractile response. The optimal concentration leading to maximumcontraction is10-4mol/l. There is no significant difference in contractionbetween the sling fibers and clasp fibers. This study indicates that the D-1-likereceptor is involved in the contractile response of the human LES.
3The selective D_2R agonist induces relaxation of the human LES, which isalso in a concentration-dependent manner. The optimal concentration leadingto maximum relaxation is10~(-3)mol/l. There is no significant difference inrelaxation between the sling fibers and clasp fibers. This study indicates thatD2R is involved in the relaxation of the human LES.
PartⅢ The contribution of dopamine receptors in the response ofhuman lower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that dopamine receptor subtypes play inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES) under the electical field stimulation (EFS), and investigate therole of dopamine receptor subtypes in vagal pathways that modulating human LES function.
Methods: Twenty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between March2011and November2011were selected in thisstudy. There were14male and6female patients, with a mean age of58years.Each specimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. The sling and clasp muscle strips wereprepared using similar methods to those we have described previously. Eachend of the muscle strips were attached with silk suture. The muscle strip wastied at one end to the electrode holder and at the other end was tied to to anisometric force transducer supported on a rack-and-pinion clamp to facilitatean accurate length adjustment of the muscle strips. Tension was recorded thesoftware of MedLab6.0. The muscle strips were mounted through concentricplatinum electrodes, makesure the distance between the end and electrode wasmore than3mm. The electrodes were connected to the output of thestimulator that delivered single pulse square wave, trains of0.5ms,50-Vsquare wave pulses at1～512Hz. Before the study, each muscle strip wasgently stretched to the the most suitable initial length, just like what we haveshown previously. The responses of muscle strips to EFS were sequentiallyassessed over a frequency range of1～512Hz, with5min between eachfrequency under control conditions. Then the muscle strip was stimulatedagain after20min of administration of selective D-1like receptor antagonistSCH23390at the concentration of10-4mol/L. The administration of selectiveD2R antagonist Spiperone hydrochloride at the concentration of10-3mol/Lwas in the same way. The responses in all of the experiments were quantifiedbased upon a percentage of the baseline value of muscle strip tone relative tothe nadir of the response. The data were expressed as means±standard error.
Results:
1Effect of the EFS on the the clasp and sling fibers of the human LES
The EFS induced a frequency-dependent relaxation in clasp fibers. Aftereach of the stimulation, there was a quick rebound contraction which was alsoin the frequency-dependent manner. The optimal frequency resulting inmaximum relaxation was64Hz. The EFS induced a frequency-dependentcontraction in sling fibers. The optimal frequency resulting in maximumcontraction was128Hz. The maximum contraction was (13.6±1.8)％.
2Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the clasp fibers of thehuman LES under the EFS
The selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent relaxation in claspfibers of the human LES induced by the EFS (F=1.142, P=0.342). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS (F=0.359, P=0.837).
3Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the sling fibers of thehuman LES under the EFS
The selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent contraction insling fibers of the human LES induced by the EFS (F=0.354, P=0.840). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent contraction in sling fibers of the human LES induced bythe EFS (F=0.651, P=0.627).
Conclusion:
1The EFS induces frequency-dependent relaxation in clasp fibers. Theoptimal frequency resulting in maximum relaxation is64Hz. EFS inducesfrequency-dependent contraction in the sling fibers. The optimal frequencyleading to maximum contraction is128Hz.
2Both of the selective D-1like dopamine receptor antagonist and theselective D_2R antagonist produces no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of clasp fiers of the human LES induced by the EFS.
3Both of the selective D-1like dopamine receptor antagonist and theselective D2R antagonist produces no significant change in thefrequency-dependent contraction in the sling fibers of the human LES inducedby the EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of sling fiers of the human LES induced by the EFS.
引文
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