肾上腺素β受体在人食管下括约肌的表达及功能研究
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摘要
食管下括约肌(Lower esophageal sphincter, LES)是位于食管胃结合部的约2-3厘米的特殊的增厚的环形肌束。1979年，Liebermann-Meffert首次提出了人食管下括约肌是由位于胃大弯侧斜行的套索纤维（sling fibers）与胃小弯侧的半环形的钩状纤维(clasp fibres)共同构成。钩状纤维，套索纤维与膈脚一起，在神经，体液等多种因素调解下，保持持续收缩状态，从而在食管胃交接部形成高压带，构成人体内生理性抗返流屏障，防止胃内容物的返流。
食管下括约肌收缩和舒张的调节机制是在中枢神经系统的支配下，由多种神经递质、激素和自身肌源性因素共同参与完成的。迷走神经的输出神经末梢和食管下括约肌的肠道运动神经元共同构成了抑制性和兴奋性运动传导通路。兴奋性运动传导通路由节前胆碱能运动神经元与节后胆碱能运动神经元共同构成。抑制性运动神经元由节前胆碱能运动神经元与节后非胆碱能非肾上腺素能神经元共同构成。兴奋性或抑制性神经通路的激活导致胆碱能节后神经元或肠道神经元多种神经递质的释放，正是通过这些释放的神经递质来实现对食管下括约肌收缩或舒张的调节。目前比较明确的是兴奋性神经传导通路的激活可以释放乙酰胆碱和P物质，从而引起食管下括约肌的收缩。抑制性传导通路的激活，引起肠道神经元释放一氧化氮和血管活性肠肽等神经递质，导致食管下括约肌的舒张。
研究表明，食管运动障碍性疾病如弥漫性食管痉挛、贲门失弛缓症以及胡桃夹食管等疾病中均表现有食管下括约肌的异常。因此，对于食管下括约肌调节机制的研究对食管运动障碍性疾病的诊断与治疗有及其重要的意义。目前国内外对于食管下括约肌的研究主要集中在一氧化氮，乙酰胆碱，血管活性肠肽以及P物质等神经递质，受体以及信号传导通路。
肾上腺素β受体属于G蛋白偶联受体家族，广泛分布于哺乳动物的中枢及外周神经系统，并发挥着极其重要的作用。肾上腺素β受体有β_1、β_2和β_3受体三种亚型。研究表明，肾上腺素β受体广泛地分布于动物的胃肠道之中。
本实验拟采用蛋白印迹法（Western-blot），逆转录聚合酶链反应(RT-PCR)，电场刺激（Electrical field stimulation, EFS）以及离体肌张力测定技术等方法，对肾上腺素β受体在人食管下括约肌的表达及功能进行研究，探讨人肾上腺素β受体在人食管下括约肌调节机制中的作用，为进一步完善人食管下括约肌的调节机制，治疗食管运动障碍性疾病提供理论依据。
第一部分肾上腺素β受体在人食管下括约肌的表达规律
目的：肾上腺素β受体是G蛋白偶联受体家族重要的受体之一。本实验采用蛋白印迹法（Western-blot），逆转录聚合酶链反应(RT-PCR)研究人食管下括约肌中的肾上腺素β受体三种亚型在套索纤维、钩状纤维、食管和胃底环形肌中的表达规律。
方法：选取河北医科大学第四医院自2011年1月到2011年10月因食管中段癌行食管大部切除术患者30例，其中男性患者24例，女性患者6例，平均年龄62岁。在手术室收集新鲜食管胃结合部标本，制备套索纤维、钩状纤维、胃底和食管环形肌肌条。提取组织总RNA，分别应用三种肾上腺素亚型的引物行RT-PCR，用Gel Pro软件分析扩增产物的光密度值(IOD)，用肾上腺素β亚型与β-actin的IOD的比值来表示肌条中mRNA的相对含量。提取组织总蛋白，将蛋白调整至相同浓度，电泳分离出肾上腺素β受体的各个亚型后转膜，再分别应用肾上腺素β受体的各个亚型抗体进行孵育，经Gel Pro软件分析各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1.8-2.0。三种β受体亚型的mRNA在各肌条均有表达：分别是β_1-AR, β_2-AR, β3-AR。β_3-AR表达量最高，它在索纤维、钩状纤维、食管平滑肌和胃底平滑肌表达水平分别为1.442±0.056；1.451±0.041；1.471±0.027；1.432±0.054；β_1-AR表达水平次之，依次为1.129±0.044；1.137±0.049；1.126±0.043；1.120±0.053；β_2-AR表达量最低，依次为0.469±0.038；0.465±0.032；0.469±0.042；0.460±0.060；在同一种肌条中，三种受体亚型的表达量有统计学差异(F=4100, P=0.00).。同一种受体亚型，在各肌条间mRNA的表达无差异(F=0.78, P=0.30)。同一种受体亚型，在各肌条间mRNA的表达无差异(F=0.78, P=0.30)。三种β受体受体亚型均有其相应蛋白条带的表达：分别是β_1-AR, β_2-AR, β_3-AR。β_3-AR表达量最高，它在套索纤维、钩状纤维、食管平滑肌和胃底平滑肌表达水平分别为0.526±0.017；0.510±0.013；0.524±0.014；0.535±0.016；β_1-AR表达水平次之，依次为0.399±0.014；0.386±0.019；0.383±0.017；0.386±0.024；β_2-AR表达量最低，依次为0.162±0.132；0.151±0.014；0.164±0.016；0.152±0.016；在同一种肌条中，三种受体亚型的表达量有统计学差异（F=39, P=0.00）。同一种受体亚型，在各肌条间蛋白的表达无差异（F=0.30, P=0.54）
结论：人LES中存在着三中肾上腺素β亚型，分别是肾上腺素β1-AR，β_2-AR，β_3-AR。其表达水平依次是β_3-AR>β_1-AR> β_2-AR。可能在人LES的功能调解中发挥着作用。
第二部分肾上腺素β受体在人食管下括约肌调节机制的作用
目的：研究非选择性的肾上腺素β受体激动剂和拮抗剂以及选择性的肾上腺素β受体激动剂和拮抗剂对人离体LES肌条的作用，探讨肾上腺素β受体在人LES收缩和舒张调节机制中的作用。
方法：选取2011年10月至2012年3月在河北医科大学第四附属医院胸三科因食管中段癌行食管大部分切除的患者共30例，其中男性20例，女性10例，平均年龄63岁。在手术室采取新鲜食管胃结合部标本立即放入4℃Krebs中，冲洗干净的标本粘膜面向上固定在盛有Krebs液体的蜡盘中，并持续通有含5％CO和95％O_2的气体。沿胃大弯侧切开标本，锐性剥离贲门部及食管下段的粘膜层及粘膜下层，发现位于食管胃交界处的发白增厚的肌环即为食管下括约肌，肉眼识别出套索纤维与钩状纤维。套索纤维位于标本的两端并呈斜行分布，而钩状纤维则位于标本的中央。游离钩状纤维和套索纤维，制备成(2~4) mm×(8~12) mm的肌条。
用丝线扎紧肌条两端，置于含Krebs液37℃恒温的10毫升浴槽中，并持续通以含5％CO和95％O_2的气体。肌条上端与JZ101型肌肉张力换能器固定，经由Medlab信号采集器记录肌条的张力变化情况。缓慢牵拉肌条使之张力稳定于200mg，此时的肌条长度即为初始长度L0.然后多次缓慢牵拉肌条，每次增加长度约为初始长度的25％左右，直至肌条长度拉伸至初长度的200％作为最适初长度。
待肌条最适初长度稳定约30分钟后，然后以浓度累计方式分别向恒温浴槽中加入非选择性的肾上腺素β受体激动剂以激活各受体亚型。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的变化情况，待张力稳定后再增加一个浓度，每次加药前都要等前一浓度达到最大反应且稳定后再加入下一浓度药物。每次加药与前一浓度稳定的时间间隔为10分钟。据此绘制累计给药的浓度-反应量效曲线。观察拮抗剂效应时，在加入激动剂前30分钟，加入拮抗剂，拮抗剂的浓度与激动剂诱导肌条产生最大效应的激动剂浓度相同。选择性的β-AR受体激动剂，选择性的β-AR拮抗剂的加药方法同前。药物诱发的肌条反应以肌条收缩或舒张的百分比的均数±标准误(x±SE)来表示。
结果
1非选择性肾上腺素β受体激动剂异丙肾上腺素在10-4mol/L的浓度可以诱导人食管下括约肌肌条产生舒张效应。应用非选择性的肾上腺素β受体阻滞剂心得安对异丙肾上腺素产生的舒张作用无效。但此作用可以被选择性肾上腺素β_3受体选择性拮抗剂L748337所阻断。
2选择性的肾上腺素β_1受体激动剂多巴酚丁胺和β_2受体激动剂沙丁胺醇对食管下括约肌肌条均没有作用。选择性的肾上腺素β_3受体激动剂BRL37344在（10~(-9)、10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)mol/L）浓度下可以诱导人食管下括约肌肌条产生浓度依赖性的舒张效应，在10-4mol/L时，肌条均达到最大收缩百分比，为（（80.4±0.6））％。应用选择性的肾上腺素β_3受体拮抗剂L748337后，BRL37344诱导肌条产生收缩的能力明显下降，肌条均达到最大收缩百分比，为（54.6±0.5）％。应用拮抗剂前后两肌条间收缩效应比较有统计学差异（P<0.05）。
结论：
1非选择性肾上腺素β受体激动剂异丙肾上腺素在10-4mol/L的浓度可以诱导人食管下括约肌肌条产生舒张效应。应用非选择性的肾上腺素β受体阻滞剂心得安对异丙肾上腺素产生的舒张作用无效。但此作用可以被肾上腺素β_3受体选择性拮抗剂L748337所阻断。提示肾上腺素β_1和β_2受体可能没有参与对人食管下括约肌的调节作用，肾上腺素β_3可能参与了对人食管下括约肌的调节。
2选择性的肾上腺素β_1受体激动剂多巴酚丁胺和β_2受体激动剂沙丁胺醇对离体食管下括约肌肌条无作用。选择性的肾上腺素β_3受体激动剂可以诱导人食管下括约肌肌条产生浓度依赖性的舒张效应，选择性的肾上腺素β_3受体拮抗剂L748337可阻断BRL37344的作用。进一步提示肾上腺素β_1和β_2受体可能没有参与对人食管下括约肌的调节作用，肾上腺素β_3可能参与了对人食管下括约肌的调节。
第三部分肾上腺素β受体在电场刺激引起的人食管下括约肌反应中的作用
目的：研究选择性肾上腺素β受体拮抗剂在电场刺激下对人食管下括约肌的作用，探讨肾上腺素β受体在人食管下括约肌舒张调节中的作用。
方法：选取2012年3月到2012年12月在河北医科大学附属第四医院胸三科因食管中上段癌，行食管大部分切除的患者30例，其中男性25例，女性5例，平均年龄60岁。食管下括约肌肌条的制备方法同前。将肌条置于37℃恒温含Krebs液的10毫升浴槽中，并持续通以含5％CO_2和95％O_2的气体。肌条的上端与肌肉张力换能器相连，下端固定于带有铂金电极的L形固定架上，利用Medlab信号采集器记录肌条的张力变化情况。肌肉张力信号经Medlab信号采集系统采集。牵拉肌肉至最适初长度方法同前。然后在持续通以含有5%CO_2和95%O_2的混合气体的Krebs液中再次温浴1小时。
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。加入阿托品后，按频率从小到大行EFS,计算刺激后产生的最大效应。刺激停止后待肌条回复平衡后，分别加入河豚毒素,选择性的肾上腺素β_1受体拮抗剂Nebivolol,选择性的β_2受体拮抗剂ICI118551和选择性的β_3受体拮抗剂L-748337，在加药20分钟后再给予EFS。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：
1EFS可以诱导食管下括约肌（钩状纤维和套索纤维），产生频率依赖性舒张反应，最大舒张电刺激频率为64HZ。EFS诱导食管下括约肌舒张的最大舒张百分比为（22.1±0.4）％。
2河豚毒素能明显降低EFS引起的人食管下括约肌频率依赖性舒张比较用药前后肌条的舒张效应有统计学差异（P<0.05）。
3EFS诱导产生的人食管下括约肌频率依赖性舒张反应，选择性β_1受体拮抗剂Nebivolol（P>0.05）和选择性的β_2受体拮抗剂ICI118551（P>0.05）对其无抑制效应，比较用药前后舒张效应无统计学意义。选择性的β_3受体拮抗剂L-748337对EFS引起的人食管下括约肌产生的频率依赖性舒张反应有抑制作用，用药前后舒张反应有统计学差异（P<0.05）。
结论：
1河豚毒素能明显降低EFS引起的人食管下括约肌频率依赖性舒张。比较用药前后肌条的舒张效应有统计学差异。提示EFS诱导产生的人食管下括约肌频率依赖性舒张是神经来源的。
2EFS诱导产生的人食管下括约肌频率依赖性舒张反应，选择性的肾上腺素β_1受体拮抗剂Nebivolol和选择性的β_2受体拮抗剂ICI118551对其无舒张效应，比较用药前后舒张效应无统计学意义。选择性的β_3受体拮抗剂L-748337对EFS引起的人食管下括约肌产生的频率依赖性舒张反应有抑制作用，用药前后舒张反应有统计学差异。提示EFS诱导产生的人食管下括约肌频率依赖性舒张可能有肾上腺素β_3受体的参与，可能没有肾上腺素β_1受体和β_2受体的参与。
The lower esophageal sphincter (LES) at the gastroesophageal junction isa special thickened circular muscle layer about2-3cm in human. In1979,Liebermann-Meffer proposed firstly that the huanm lower esophagealsphincter consists of sling fibers at the greater curvature and clasp fibers at thelesser curvature. The clasp fibers, the sling fibers and the crural diaphragmregulated with the mediation of the nervers, body fluids and other factors,maintain a continued contraction, which forms a high pressure band atesophagogastric and constitute the physilolgical antireflux barrier to preventthe reflux of the gastric contents into the esophagus.
The adjustment mechanism of contraction and relaxation of the LES isrugulated by several neurotransmitters, hormones and spontaneous myogenicfactors in the control of the central nervous system. The output nerve endingof the vagus nerve and the intestinal motor neurons of the lower esophagealsphincter constitute the inhibitory and excitatory movement pathway. Theexcitatory movement pathway consists of preganglionic cholinergic sportsneurons and postganglionic cholinergic motor neurons and the inhibitorymovement pathway consists of preganglionic cholinergic motor neurons andpostganglionic non-cholinergic non-adrenergic neurons. Activation ofexcitatory or inhibitory neural pathway leads to the release of variety ofneurotransmitte of cholinergic postganglionic neurons or intestinal neurons,which could adjust the contraction or relaxation of the lower esophagealsphincter. Now it is obvious that the activation of the excitatoryneurotransmitter pathway could release acetylcholine and substance P, whichlead to the contraction of the lower esophageal sphincter. The activation of theinhibitory neurotransmitter pathway could release nitric oxide and vasoactiveintestinal peptide, which lead to the relaxation of the lower esophageal sphincter.
It is demonstrated that esophageal motor disorders such as diffuseesophageal spasm, achalasia and nutcracker esophagus showed abnormalitiesof the lower esophageal sphincter. Therefore, the study of the regulationmechanism of the lower esophageal sphincter is important for the diagnosisand treatment of esophageal motility disorders. The studies showed that theregulatory mechanism of the LES involves of Nitric oxide, acetylcholine,vasoactive intestinal peptide, their receptors snd signal transduction pathways.
β-adrenergic receptor is a member of the G protein-coupled receptorfamily. It has been demonstrated that there are three receptor subtypesincluding β_1-AR, β_2-AR, β_3-AR, which are widely present in themammalian central and peripheral nervous system, such as brain,cardiovascular system and gastrointestinal tract, and are importantphysiological modulator
By western blotting, reverse transcription-polymerase chain reaction (RT-PCR), measurement of muscle tension in vitro and electrical field stimulation(EFS)，the purpose of this thesis is to investigateβ-AR expression andfunction of the lower esophageal sphincter and to investigate the β-AR inhuman esophageal sphincter adjustment mechanism, which could furtherimprove the human lower esophageal sphincter regulating mechanisms andprovide a theoretical basis for the treatment of esophageal motility disorders.
Part Ⅰ Expression of β-adrenergic receptor in the human loweresophageal sphincter
Objective:β-adrenergic receptor is a member of the G protein-coupledreceptor family. In the present study, we identified the expression of mRNAand protein of β-AR receptors in four muscle strips including sling fibers,clasp fibers, circular muscle strips of the esophagus and stomach by westernblotting, reverse transcription-polymerase chain reaction (RT-PCR).
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in the Department of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from January2011to October2011. There were21males and9females, with an average age of64years (ranged55to68). In the laboratory,fresh esophagogastric junction specimens collected in the operating room wereprepared into the sling fibers, clasp fibers and circular muscle strips ofesophagus and stomach. Total RNA was extracted. Reversetranscription-polymerase chain reaction (RT-PCR) was performed usingprimers designed specifically to match the β-AR mRNA. PCR productswere determined by using the Gel-Pro gel imaging analysis system.Densitometry for bands on PCR products was determined by imaging software.The relative expression level of each gene was normalized by the value ofβ-actin. Total proteins were extracted from muscle strips by using proteinextraction kit. Proteins were adjusted to the same concentration. The threesubtypes were separated by electrophoresis and transferred onto apolyvinylidene difluoride (PVDF). The detection of the protein expressionwas operated using different β-AR polyclonal antibody. The membrane wasdetected by infrared fluorescence imaging instrument. The relative expressionlevel of each protein was normalized by the value of β-actin..
The value of A260/280of total RNA was between1.8and2.0afterultraviolet spectrophotometry. The band of β-actin mRNA was uniformly540bp. Transcripts for β_1-AR, β_2-AR, and β_3-AR were identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent β-AR mRNA in the same muscle strips (F=4100, P=0.00). Therank order of the extent of expression was β_3-AR> β_1-AR> β_2-AR.However, there was no significant difference in mRNA expression of β-ARbetween the four muscle strips(F=0.78, P=0.30). β_1-AR, β_2-AR, andβ3-AR protein expression were identified. There was a significant difference inIOD values for different β-AR in the same muscle strip (F=39, P=0.00).The rank order of the value was the same as the result of the RT-PCR. Therewas no significant difference in IOD values between the four muscle strips。
Conclusion: β_1-AR, β_2-AR,β_3-AR can be detected in the humanLES, the extent of expression in secending order is β_3-AR>β_1-AR>β_2-AR,and probably contribut to LES function.
Part Ⅱ The role of β-adrenoceptors in modulating human loweresophageal sphincter
Objective： To study the effects of selectinve and nonselective β-adrenoceptors agonists and antagonists on the LES muscle strips，andinvestigate the role of β-adrenoceptors subtypes in adjusting contraction andrelaxation of the LES.
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from October2011to March2012. There were20males and10females, with an average age of63years. In the laboratory, freshesophagogastric junction specimens collected in the operating room wereimmediately placed in the4℃Krebs. After washed with37℃Krebssolution, specimens were pined on a wax plate containing TBS, withcontinuous mixed gas of95%O_2and5%CO_2. The mucosa and submucosawere then gently removed by sharp dissection. The gastric sling and claspfibers could be identified as thickened bands of circular oriented smoothmuscle in the gastric cardia, adjacent to the greater and lesser curvature of thestomach, respectively. The sling and clasp muscle strips were prepared usingthe method that we have described previously. The sling fibers, clasp fiberswere separated and prepared into (2~4) mm×(8~12) mm muscle strips.
The ends of the muscle strips were tied with silk, placed in a10ml bathcontaining Krebs solution maintained37℃，with continuous mixed gas of95%O_2and5%CO_2. The upper end of the muscle strips was fixed with JZ101muscle tension transducer and the changes of the muscle strips tension wasrecord by the Medlab signal acquisition system. The muscle strips were stretchslowly and make the tension maintaining at200mg, and the length of the muscle strips at that tension is the initial length L0. The muscle strips werestretch slowly and repeatedly, with each about25%of the initial length, untilthe muscle strips were stretched to200%of the initial length which is as theoptimum initial length.
The muscle strips with the optimum initial were stabilized for30minutes, then the non-selective agonists of the β-adrenoceptors subtypewere added into the bath with a cumulative manner from10~(-9) to10~(-3) mol/Lrespectively. Successive concentrations of the agonists were not added untilthe response of the previous concentration stabilized. Ten minutes betweenadditions were allowed for lack of effect. Based on the above results, we drawthe cumulative administration concentration-response dose-response curves.The antagonist was added before adding the agonist for observed the effect ofthe antagonist and the concentration of the antagonist is same to theconcentration of the agonist that induced the muscle strips to produce themaximum effect. The administration of selectiveβ-adrenoceptors agonist andselectiveβ-adrenoceptors antagonist were in the same way. The responses inall of the experiments were quantified based upon a percentage of the baselinevalue of muscle strip tone relative to the nadir of the response. The data wasexpressed as means±standard error.
Results:
1Isoproterenol, a non-selective β-adrenoceptor agonist, could inducerelaxation effect of the lower esophageal sphincter muscle strips at theconcentration of10~(-4)mol/L. Propranolol, non-selective β-adrenoceptorsantagonists was no effect on the relaxation effect induced by Isoproterenol，butwhich could be blocked by L748337, a selective β-adrenoceptor agonist.
2Dobutamine, a selectiveβ_1-adrenoceptor agonist, and salbutamol, aselective β_2-adrenoceptor agonist, had no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,could induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect at the concentration from10~(-9)to10~(-3)mol/L. The optimal concentration leading to maximum contraction was 10~(-4)mol/L. The maximum relaxation of the LES muscle strips was(80±0.6)％. L748337, a selectiveβ_3-adrenoceptor antagonist could block theeffect of BRL37344.
Conclusion:
1soproterenol, a non-selective β-adrenoceptor, induces relaxationeffect of the lower esophageal sphincter muscle strips at the concentration of10~(-4)mol/L. Propranolol, non-selective β-adrenoceptors antagonists has noeffect on the relaxation effect induced by Isoproterenol，but can be blocked byL748337, a selective β-adrenoceptor agonist. It implys that the β3-adrenoceptor other thanβ_1-and β_2-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.
2Dobutamine, a selectiveβ_1-adrenoceptor agonist, and salbutamol,and a selectiveβ_2-adrenoceptor agonist, has no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect. L748337, a selective β3-adrenoceptor antagonist could block the effect of BRL37344. The β3-adrenoceptor other than β_1-and β_2-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.
PartⅢ The contribution of β-adrenoceptor in the response of humanlower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that the β-adrenoceptor antagnostsplay in regulating human lower esophageal sphincter (LES) under the electicalfield stimulation (EFS), and investigate the role of β-adrenoceptos inmodulating human LES function.
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from March2012to December2012. There were25males and5females, with an average age of60years. The LES muscle strips were prepared using similar methods to that we have described previously. Themuscle strips were placed in a10ml bath containing Krebs solution maintained37℃，with continuous mixed gas of95%O_2and5%CO_2. The upper end ofthe muscle strips were fixed with muscle tension transducer and the lower endwas connected to the L-shaped bracket with a platinum electrode, with usingof the Medlab signal acquisition to record muscle strip tension changes. Themuscle strips were stretched to optimum initial length using similar methodsto that we have described previously and had a warm bath for1hour in Krebssolution with continuous mixed gas of95%O_2and5%CO_2.
EFS stimulation parameters: single-pulse square wave, pulse width5ms,voltage50V, frequency1-512Hz. After added atropine, the muscle strips issubjected to electrical stimulation according to the frequency from small to bigand the maximum effect to EFS was assessed. The muscle strip was stimulatedagain after20min of administration of Tetrodotoxin, Nebivolol (a selectiveβ1-adrenoceptor antagonists), ICI118551(a selective β_2-adrenoceptorantagonist) and L-748337(a selective β_3-adrenoceptor antagonist). Theresponses in all of the experiments were quantified based upon a percentage ofthe baseline value of muscle strip tone relative to the nadir of the response.The data was expressed as means±standard error.
Results:
1The EFS could induce the lower esophageal sphincter (clasp fibers andsling fibers) to produce the frequency dependent of the relaxation response,and the maximum diastolic electrical stimulation frequency was64HZ. Themaximum relaxation percentage of the lower esophageal sphincter byEFS-induced was (22.1±0.4)%.
2Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration.
3Nebivolol and ICI118551had no effect on the relaxation of the LESmuscle strips by EFS-induced. There was significant no difference before and after administration. L-748337could inhibited the the frequency dependenceof the relaxation response of the LES by EFS-induced. There was significantdifference before and after administration.
Conclusion:
1Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration. It is prompted that the frequency-dependent relaxation oflower esophageal sphincter by EFS-induced is neurogenic in origin.
2Nebivolol and ICI118551have no effect on the relaxation of the LESmuscle strips by EFS-induced. The frequency-dependent contraction in thesling fibers of the human LES can be induced by the EFS. The β3-adrenoceptor other thanβ_1-and β_2-adrenoceptor may be involved in thefrequency-dependent relaxation of the human LES induced by the EFS.
食管下括约肌收缩和舒张的调节机制是在中枢神经系统的支配下，由多种神经递质、激素和自身肌源性因素共同参与完成的。迷走神经的输出神经末梢和食管下括约肌的肠道运动神经元共同构成了抑制性和兴奋性运动传导通路。兴奋性运动传导通路由节前胆碱能运动神经元与节后胆碱能运动神经元共同构成。抑制性运动神经元由节前胆碱能运动神经元与节后非胆碱能非肾上腺素能神经元共同构成。兴奋性或抑制性神经通路的激活导致胆碱能节后神经元或肠道神经元多种神经递质的释放，正是通过这些释放的神经递质来实现对食管下括约肌收缩或舒张的调节。目前比较明确的是兴奋性神经传导通路的激活可以释放乙酰胆碱和P物质，从而引起食管下括约肌的收缩。抑制性传导通路的激活，引起肠道神经元释放一氧化氮和血管活性肠肽等神经递质，导致食管下括约肌的舒张。
研究表明，食管运动障碍性疾病如弥漫性食管痉挛、贲门失弛缓症以及胡桃夹食管等疾病中均表现有食管下括约肌的异常。因此，对于食管下括约肌调节机制的研究对食管运动障碍性疾病的诊断与治疗有及其重要的意义。目前国内外对于食管下括约肌的研究主要集中在一氧化氮，乙酰胆碱，血管活性肠肽以及P物质等神经递质，受体以及信号传导通路。
肾上腺素β受体属于G蛋白偶联受体家族，广泛分布于哺乳动物的中枢及外周神经系统，并发挥着极其重要的作用。肾上腺素β受体有β_1、β_2和β_3受体三种亚型。研究表明，肾上腺素β受体广泛地分布于动物的胃肠道之中。
本实验拟采用蛋白印迹法（Western-blot），逆转录聚合酶链反应(RT-PCR)，电场刺激（Electrical field stimulation, EFS）以及离体肌张力测定技术等方法，对肾上腺素β受体在人食管下括约肌的表达及功能进行研究，探讨人肾上腺素β受体在人食管下括约肌调节机制中的作用，为进一步完善人食管下括约肌的调节机制，治疗食管运动障碍性疾病提供理论依据。
第一部分肾上腺素β受体在人食管下括约肌的表达规律
目的：肾上腺素β受体是G蛋白偶联受体家族重要的受体之一。本实验采用蛋白印迹法（Western-blot），逆转录聚合酶链反应(RT-PCR)研究人食管下括约肌中的肾上腺素β受体三种亚型在套索纤维、钩状纤维、食管和胃底环形肌中的表达规律。
方法：选取河北医科大学第四医院自2011年1月到2011年10月因食管中段癌行食管大部切除术患者30例，其中男性患者24例，女性患者6例，平均年龄62岁。在手术室收集新鲜食管胃结合部标本，制备套索纤维、钩状纤维、胃底和食管环形肌肌条。提取组织总RNA，分别应用三种肾上腺素亚型的引物行RT-PCR，用Gel Pro软件分析扩增产物的光密度值(IOD)，用肾上腺素β亚型与β-actin的IOD的比值来表示肌条中mRNA的相对含量。提取组织总蛋白，将蛋白调整至相同浓度，电泳分离出肾上腺素β受体的各个亚型后转膜，再分别应用肾上腺素β受体的各个亚型抗体进行孵育，经Gel Pro软件分析各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1.8-2.0。三种β受体亚型的mRNA在各肌条均有表达：分别是β_1-AR, β_2-AR, β3-AR。β_3-AR表达量最高，它在索纤维、钩状纤维、食管平滑肌和胃底平滑肌表达水平分别为1.442±0.056；1.451±0.041；1.471±0.027；1.432±0.054；β_1-AR表达水平次之，依次为1.129±0.044；1.137±0.049；1.126±0.043；1.120±0.053；β_2-AR表达量最低，依次为0.469±0.038；0.465±0.032；0.469±0.042；0.460±0.060；在同一种肌条中，三种受体亚型的表达量有统计学差异(F=4100, P=0.00).。同一种受体亚型，在各肌条间mRNA的表达无差异(F=0.78, P=0.30)。同一种受体亚型，在各肌条间mRNA的表达无差异(F=0.78, P=0.30)。三种β受体受体亚型均有其相应蛋白条带的表达：分别是β_1-AR, β_2-AR, β_3-AR。β_3-AR表达量最高，它在套索纤维、钩状纤维、食管平滑肌和胃底平滑肌表达水平分别为0.526±0.017；0.510±0.013；0.524±0.014；0.535±0.016；β_1-AR表达水平次之，依次为0.399±0.014；0.386±0.019；0.383±0.017；0.386±0.024；β_2-AR表达量最低，依次为0.162±0.132；0.151±0.014；0.164±0.016；0.152±0.016；在同一种肌条中，三种受体亚型的表达量有统计学差异（F=39, P=0.00）。同一种受体亚型，在各肌条间蛋白的表达无差异（F=0.30, P=0.54）
结论：人LES中存在着三中肾上腺素β亚型，分别是肾上腺素β1-AR，β_2-AR，β_3-AR。其表达水平依次是β_3-AR>β_1-AR> β_2-AR。可能在人LES的功能调解中发挥着作用。
第二部分肾上腺素β受体在人食管下括约肌调节机制的作用
目的：研究非选择性的肾上腺素β受体激动剂和拮抗剂以及选择性的肾上腺素β受体激动剂和拮抗剂对人离体LES肌条的作用，探讨肾上腺素β受体在人LES收缩和舒张调节机制中的作用。
方法：选取2011年10月至2012年3月在河北医科大学第四附属医院胸三科因食管中段癌行食管大部分切除的患者共30例，其中男性20例，女性10例，平均年龄63岁。在手术室采取新鲜食管胃结合部标本立即放入4℃Krebs中，冲洗干净的标本粘膜面向上固定在盛有Krebs液体的蜡盘中，并持续通有含5％CO和95％O_2的气体。沿胃大弯侧切开标本，锐性剥离贲门部及食管下段的粘膜层及粘膜下层，发现位于食管胃交界处的发白增厚的肌环即为食管下括约肌，肉眼识别出套索纤维与钩状纤维。套索纤维位于标本的两端并呈斜行分布，而钩状纤维则位于标本的中央。游离钩状纤维和套索纤维，制备成(2~4) mm×(8~12) mm的肌条。
用丝线扎紧肌条两端，置于含Krebs液37℃恒温的10毫升浴槽中，并持续通以含5％CO和95％O_2的气体。肌条上端与JZ101型肌肉张力换能器固定，经由Medlab信号采集器记录肌条的张力变化情况。缓慢牵拉肌条使之张力稳定于200mg，此时的肌条长度即为初始长度L0.然后多次缓慢牵拉肌条，每次增加长度约为初始长度的25％左右，直至肌条长度拉伸至初长度的200％作为最适初长度。
待肌条最适初长度稳定约30分钟后，然后以浓度累计方式分别向恒温浴槽中加入非选择性的肾上腺素β受体激动剂以激活各受体亚型。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的变化情况，待张力稳定后再增加一个浓度，每次加药前都要等前一浓度达到最大反应且稳定后再加入下一浓度药物。每次加药与前一浓度稳定的时间间隔为10分钟。据此绘制累计给药的浓度-反应量效曲线。观察拮抗剂效应时，在加入激动剂前30分钟，加入拮抗剂，拮抗剂的浓度与激动剂诱导肌条产生最大效应的激动剂浓度相同。选择性的β-AR受体激动剂，选择性的β-AR拮抗剂的加药方法同前。药物诱发的肌条反应以肌条收缩或舒张的百分比的均数±标准误(x±SE)来表示。
结果
1非选择性肾上腺素β受体激动剂异丙肾上腺素在10-4mol/L的浓度可以诱导人食管下括约肌肌条产生舒张效应。应用非选择性的肾上腺素β受体阻滞剂心得安对异丙肾上腺素产生的舒张作用无效。但此作用可以被选择性肾上腺素β_3受体选择性拮抗剂L748337所阻断。
2选择性的肾上腺素β_1受体激动剂多巴酚丁胺和β_2受体激动剂沙丁胺醇对食管下括约肌肌条均没有作用。选择性的肾上腺素β_3受体激动剂BRL37344在（10~(-9)、10~(-8)、10~(-7)、10~(-6)、10~(-5)、10~(-4)mol/L）浓度下可以诱导人食管下括约肌肌条产生浓度依赖性的舒张效应，在10-4mol/L时，肌条均达到最大收缩百分比，为（（80.4±0.6））％。应用选择性的肾上腺素β_3受体拮抗剂L748337后，BRL37344诱导肌条产生收缩的能力明显下降，肌条均达到最大收缩百分比，为（54.6±0.5）％。应用拮抗剂前后两肌条间收缩效应比较有统计学差异（P<0.05）。
结论：
1非选择性肾上腺素β受体激动剂异丙肾上腺素在10-4mol/L的浓度可以诱导人食管下括约肌肌条产生舒张效应。应用非选择性的肾上腺素β受体阻滞剂心得安对异丙肾上腺素产生的舒张作用无效。但此作用可以被肾上腺素β_3受体选择性拮抗剂L748337所阻断。提示肾上腺素β_1和β_2受体可能没有参与对人食管下括约肌的调节作用，肾上腺素β_3可能参与了对人食管下括约肌的调节。
2选择性的肾上腺素β_1受体激动剂多巴酚丁胺和β_2受体激动剂沙丁胺醇对离体食管下括约肌肌条无作用。选择性的肾上腺素β_3受体激动剂可以诱导人食管下括约肌肌条产生浓度依赖性的舒张效应，选择性的肾上腺素β_3受体拮抗剂L748337可阻断BRL37344的作用。进一步提示肾上腺素β_1和β_2受体可能没有参与对人食管下括约肌的调节作用，肾上腺素β_3可能参与了对人食管下括约肌的调节。
第三部分肾上腺素β受体在电场刺激引起的人食管下括约肌反应中的作用
目的：研究选择性肾上腺素β受体拮抗剂在电场刺激下对人食管下括约肌的作用，探讨肾上腺素β受体在人食管下括约肌舒张调节中的作用。
方法：选取2012年3月到2012年12月在河北医科大学附属第四医院胸三科因食管中上段癌，行食管大部分切除的患者30例，其中男性25例，女性5例，平均年龄60岁。食管下括约肌肌条的制备方法同前。将肌条置于37℃恒温含Krebs液的10毫升浴槽中，并持续通以含5％CO_2和95％O_2的气体。肌条的上端与肌肉张力换能器相连，下端固定于带有铂金电极的L形固定架上，利用Medlab信号采集器记录肌条的张力变化情况。肌肉张力信号经Medlab信号采集系统采集。牵拉肌肉至最适初长度方法同前。然后在持续通以含有5%CO_2和95%O_2的混合气体的Krebs液中再次温浴1小时。
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。加入阿托品后，按频率从小到大行EFS,计算刺激后产生的最大效应。刺激停止后待肌条回复平衡后，分别加入河豚毒素,选择性的肾上腺素β_1受体拮抗剂Nebivolol,选择性的β_2受体拮抗剂ICI118551和选择性的β_3受体拮抗剂L-748337，在加药20分钟后再给予EFS。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：
1EFS可以诱导食管下括约肌（钩状纤维和套索纤维），产生频率依赖性舒张反应，最大舒张电刺激频率为64HZ。EFS诱导食管下括约肌舒张的最大舒张百分比为（22.1±0.4）％。
2河豚毒素能明显降低EFS引起的人食管下括约肌频率依赖性舒张比较用药前后肌条的舒张效应有统计学差异（P<0.05）。
3EFS诱导产生的人食管下括约肌频率依赖性舒张反应，选择性β_1受体拮抗剂Nebivolol（P>0.05）和选择性的β_2受体拮抗剂ICI118551（P>0.05）对其无抑制效应，比较用药前后舒张效应无统计学意义。选择性的β_3受体拮抗剂L-748337对EFS引起的人食管下括约肌产生的频率依赖性舒张反应有抑制作用，用药前后舒张反应有统计学差异（P<0.05）。
结论：
1河豚毒素能明显降低EFS引起的人食管下括约肌频率依赖性舒张。比较用药前后肌条的舒张效应有统计学差异。提示EFS诱导产生的人食管下括约肌频率依赖性舒张是神经来源的。
2EFS诱导产生的人食管下括约肌频率依赖性舒张反应，选择性的肾上腺素β_1受体拮抗剂Nebivolol和选择性的β_2受体拮抗剂ICI118551对其无舒张效应，比较用药前后舒张效应无统计学意义。选择性的β_3受体拮抗剂L-748337对EFS引起的人食管下括约肌产生的频率依赖性舒张反应有抑制作用，用药前后舒张反应有统计学差异。提示EFS诱导产生的人食管下括约肌频率依赖性舒张可能有肾上腺素β_3受体的参与，可能没有肾上腺素β_1受体和β_2受体的参与。
The lower esophageal sphincter (LES) at the gastroesophageal junction isa special thickened circular muscle layer about2-3cm in human. In1979,Liebermann-Meffer proposed firstly that the huanm lower esophagealsphincter consists of sling fibers at the greater curvature and clasp fibers at thelesser curvature. The clasp fibers, the sling fibers and the crural diaphragmregulated with the mediation of the nervers, body fluids and other factors,maintain a continued contraction, which forms a high pressure band atesophagogastric and constitute the physilolgical antireflux barrier to preventthe reflux of the gastric contents into the esophagus.
The adjustment mechanism of contraction and relaxation of the LES isrugulated by several neurotransmitters, hormones and spontaneous myogenicfactors in the control of the central nervous system. The output nerve endingof the vagus nerve and the intestinal motor neurons of the lower esophagealsphincter constitute the inhibitory and excitatory movement pathway. Theexcitatory movement pathway consists of preganglionic cholinergic sportsneurons and postganglionic cholinergic motor neurons and the inhibitorymovement pathway consists of preganglionic cholinergic motor neurons andpostganglionic non-cholinergic non-adrenergic neurons. Activation ofexcitatory or inhibitory neural pathway leads to the release of variety ofneurotransmitte of cholinergic postganglionic neurons or intestinal neurons,which could adjust the contraction or relaxation of the lower esophagealsphincter. Now it is obvious that the activation of the excitatoryneurotransmitter pathway could release acetylcholine and substance P, whichlead to the contraction of the lower esophageal sphincter. The activation of theinhibitory neurotransmitter pathway could release nitric oxide and vasoactiveintestinal peptide, which lead to the relaxation of the lower esophageal sphincter.
It is demonstrated that esophageal motor disorders such as diffuseesophageal spasm, achalasia and nutcracker esophagus showed abnormalitiesof the lower esophageal sphincter. Therefore, the study of the regulationmechanism of the lower esophageal sphincter is important for the diagnosisand treatment of esophageal motility disorders. The studies showed that theregulatory mechanism of the LES involves of Nitric oxide, acetylcholine,vasoactive intestinal peptide, their receptors snd signal transduction pathways.
β-adrenergic receptor is a member of the G protein-coupled receptorfamily. It has been demonstrated that there are three receptor subtypesincluding β_1-AR, β_2-AR, β_3-AR, which are widely present in themammalian central and peripheral nervous system, such as brain,cardiovascular system and gastrointestinal tract, and are importantphysiological modulator
By western blotting, reverse transcription-polymerase chain reaction (RT-PCR), measurement of muscle tension in vitro and electrical field stimulation(EFS)，the purpose of this thesis is to investigateβ-AR expression andfunction of the lower esophageal sphincter and to investigate the β-AR inhuman esophageal sphincter adjustment mechanism, which could furtherimprove the human lower esophageal sphincter regulating mechanisms andprovide a theoretical basis for the treatment of esophageal motility disorders.
Part Ⅰ Expression of β-adrenergic receptor in the human loweresophageal sphincter
Objective:β-adrenergic receptor is a member of the G protein-coupledreceptor family. In the present study, we identified the expression of mRNAand protein of β-AR receptors in four muscle strips including sling fibers,clasp fibers, circular muscle strips of the esophagus and stomach by westernblotting, reverse transcription-polymerase chain reaction (RT-PCR).
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in the Department of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from January2011to October2011. There were21males and9females, with an average age of64years (ranged55to68). In the laboratory,fresh esophagogastric junction specimens collected in the operating room wereprepared into the sling fibers, clasp fibers and circular muscle strips ofesophagus and stomach. Total RNA was extracted. Reversetranscription-polymerase chain reaction (RT-PCR) was performed usingprimers designed specifically to match the β-AR mRNA. PCR productswere determined by using the Gel-Pro gel imaging analysis system.Densitometry for bands on PCR products was determined by imaging software.The relative expression level of each gene was normalized by the value ofβ-actin. Total proteins were extracted from muscle strips by using proteinextraction kit. Proteins were adjusted to the same concentration. The threesubtypes were separated by electrophoresis and transferred onto apolyvinylidene difluoride (PVDF). The detection of the protein expressionwas operated using different β-AR polyclonal antibody. The membrane wasdetected by infrared fluorescence imaging instrument. The relative expressionlevel of each protein was normalized by the value of β-actin..
The value of A260/280of total RNA was between1.8and2.0afterultraviolet spectrophotometry. The band of β-actin mRNA was uniformly540bp. Transcripts for β_1-AR, β_2-AR, and β_3-AR were identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent β-AR mRNA in the same muscle strips (F=4100, P=0.00). Therank order of the extent of expression was β_3-AR> β_1-AR> β_2-AR.However, there was no significant difference in mRNA expression of β-ARbetween the four muscle strips(F=0.78, P=0.30). β_1-AR, β_2-AR, andβ3-AR protein expression were identified. There was a significant difference inIOD values for different β-AR in the same muscle strip (F=39, P=0.00).The rank order of the value was the same as the result of the RT-PCR. Therewas no significant difference in IOD values between the four muscle strips。
Conclusion: β_1-AR, β_2-AR,β_3-AR can be detected in the humanLES, the extent of expression in secending order is β_3-AR>β_1-AR>β_2-AR,and probably contribut to LES function.
Part Ⅱ The role of β-adrenoceptors in modulating human loweresophageal sphincter
Objective： To study the effects of selectinve and nonselective β-adrenoceptors agonists and antagonists on the LES muscle strips，andinvestigate the role of β-adrenoceptors subtypes in adjusting contraction andrelaxation of the LES.
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from October2011to March2012. There were20males and10females, with an average age of63years. In the laboratory, freshesophagogastric junction specimens collected in the operating room wereimmediately placed in the4℃Krebs. After washed with37℃Krebssolution, specimens were pined on a wax plate containing TBS, withcontinuous mixed gas of95%O_2and5%CO_2. The mucosa and submucosawere then gently removed by sharp dissection. The gastric sling and claspfibers could be identified as thickened bands of circular oriented smoothmuscle in the gastric cardia, adjacent to the greater and lesser curvature of thestomach, respectively. The sling and clasp muscle strips were prepared usingthe method that we have described previously. The sling fibers, clasp fiberswere separated and prepared into (2~4) mm×(8~12) mm muscle strips.
The ends of the muscle strips were tied with silk, placed in a10ml bathcontaining Krebs solution maintained37℃，with continuous mixed gas of95%O_2and5%CO_2. The upper end of the muscle strips was fixed with JZ101muscle tension transducer and the changes of the muscle strips tension wasrecord by the Medlab signal acquisition system. The muscle strips were stretchslowly and make the tension maintaining at200mg, and the length of the muscle strips at that tension is the initial length L0. The muscle strips werestretch slowly and repeatedly, with each about25%of the initial length, untilthe muscle strips were stretched to200%of the initial length which is as theoptimum initial length.
The muscle strips with the optimum initial were stabilized for30minutes, then the non-selective agonists of the β-adrenoceptors subtypewere added into the bath with a cumulative manner from10~(-9) to10~(-3) mol/Lrespectively. Successive concentrations of the agonists were not added untilthe response of the previous concentration stabilized. Ten minutes betweenadditions were allowed for lack of effect. Based on the above results, we drawthe cumulative administration concentration-response dose-response curves.The antagonist was added before adding the agonist for observed the effect ofthe antagonist and the concentration of the antagonist is same to theconcentration of the agonist that induced the muscle strips to produce themaximum effect. The administration of selectiveβ-adrenoceptors agonist andselectiveβ-adrenoceptors antagonist were in the same way. The responses inall of the experiments were quantified based upon a percentage of the baselinevalue of muscle strip tone relative to the nadir of the response. The data wasexpressed as means±standard error.
Results:
1Isoproterenol, a non-selective β-adrenoceptor agonist, could inducerelaxation effect of the lower esophageal sphincter muscle strips at theconcentration of10~(-4)mol/L. Propranolol, non-selective β-adrenoceptorsantagonists was no effect on the relaxation effect induced by Isoproterenol，butwhich could be blocked by L748337, a selective β-adrenoceptor agonist.
2Dobutamine, a selectiveβ_1-adrenoceptor agonist, and salbutamol, aselective β_2-adrenoceptor agonist, had no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,could induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect at the concentration from10~(-9)to10~(-3)mol/L. The optimal concentration leading to maximum contraction was 10~(-4)mol/L. The maximum relaxation of the LES muscle strips was(80±0.6)％. L748337, a selectiveβ_3-adrenoceptor antagonist could block theeffect of BRL37344.
Conclusion:
1soproterenol, a non-selective β-adrenoceptor, induces relaxationeffect of the lower esophageal sphincter muscle strips at the concentration of10~(-4)mol/L. Propranolol, non-selective β-adrenoceptors antagonists has noeffect on the relaxation effect induced by Isoproterenol，but can be blocked byL748337, a selective β-adrenoceptor agonist. It implys that the β3-adrenoceptor other thanβ_1-and β_2-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.
2Dobutamine, a selectiveβ_1-adrenoceptor agonist, and salbutamol,and a selectiveβ_2-adrenoceptor agonist, has no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect. L748337, a selective β3-adrenoceptor antagonist could block the effect of BRL37344. The β3-adrenoceptor other than β_1-and β_2-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.
PartⅢ The contribution of β-adrenoceptor in the response of humanlower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that the β-adrenoceptor antagnostsplay in regulating human lower esophageal sphincter (LES) under the electicalfield stimulation (EFS), and investigate the role of β-adrenoceptos inmodulating human LES function.
Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from March2012to December2012. There were25males and5females, with an average age of60years. The LES muscle strips were prepared using similar methods to that we have described previously. Themuscle strips were placed in a10ml bath containing Krebs solution maintained37℃，with continuous mixed gas of95%O_2and5%CO_2. The upper end ofthe muscle strips were fixed with muscle tension transducer and the lower endwas connected to the L-shaped bracket with a platinum electrode, with usingof the Medlab signal acquisition to record muscle strip tension changes. Themuscle strips were stretched to optimum initial length using similar methodsto that we have described previously and had a warm bath for1hour in Krebssolution with continuous mixed gas of95%O_2and5%CO_2.
EFS stimulation parameters: single-pulse square wave, pulse width5ms,voltage50V, frequency1-512Hz. After added atropine, the muscle strips issubjected to electrical stimulation according to the frequency from small to bigand the maximum effect to EFS was assessed. The muscle strip was stimulatedagain after20min of administration of Tetrodotoxin, Nebivolol (a selectiveβ1-adrenoceptor antagonists), ICI118551(a selective β_2-adrenoceptorantagonist) and L-748337(a selective β_3-adrenoceptor antagonist). Theresponses in all of the experiments were quantified based upon a percentage ofthe baseline value of muscle strip tone relative to the nadir of the response.The data was expressed as means±standard error.
Results:
1The EFS could induce the lower esophageal sphincter (clasp fibers andsling fibers) to produce the frequency dependent of the relaxation response,and the maximum diastolic electrical stimulation frequency was64HZ. Themaximum relaxation percentage of the lower esophageal sphincter byEFS-induced was (22.1±0.4)%.
2Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration.
3Nebivolol and ICI118551had no effect on the relaxation of the LESmuscle strips by EFS-induced. There was significant no difference before and after administration. L-748337could inhibited the the frequency dependenceof the relaxation response of the LES by EFS-induced. There was significantdifference before and after administration.
Conclusion:
1Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration. It is prompted that the frequency-dependent relaxation oflower esophageal sphincter by EFS-induced is neurogenic in origin.
2Nebivolol and ICI118551have no effect on the relaxation of the LESmuscle strips by EFS-induced. The frequency-dependent contraction in thesling fibers of the human LES can be induced by the EFS. The β3-adrenoceptor other thanβ_1-and β_2-adrenoceptor may be involved in thefrequency-dependent relaxation of the human LES induced by the EFS.
引文
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