溶血磷脂酸受体在人食管下括约肌的表达及功能研究
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摘要
食管胃结合部（Esophagogastric junction, EGJ）是一个复杂的瓣膜结构，其主要是由食管下括约肌（Lower esophageal sphincter, LES）和膈肌脚组成的高压带。LES是大约2-3厘米的增厚的环形肌肉束，并在1979年由Liebermann-Meffert第一次提出来的，其是由胃小弯侧半环形的钩状纤维和胃大弯侧的斜行的套索纤维共同构成的。在非吞咽时防止胃内容物反流，而在吞咽过程中保持开放并促进食管或胃内容物向下流动。
LES发生的收缩和舒张反应的调节机制是在中枢神经系统的支配下，由其自身肌源性因素、多种激素和神经递质参与而共同完成的。LES的收缩和舒张是由迷走神经节前纤维和交感神经节后纤维支配的。迷走神经节前纤维不仅可以通过肌间神经丛并且也可以通过抑制性神经通路和兴奋性神经通路来完成对LES的舒张和收缩功能的支配。其抑制性和兴奋性节前细胞分别位于迷走神经背侧运动核的尾部和前部。典型的是，胆碱能神经元是迷走神经节前纤维通过烟碱和毒蕈碱受体发挥其对肌间神经丛的抑制作用。兴奋性和抑制性神经元通路通过影响肌源性张力来影响LES张力。兴奋性肌间神经元通过释放乙酰胆碱和P物质增加LES张力。而抑制性肌间神经元是通过产生NO来减少LES的张力，NO是最主要的抑制性神经递质，在吞咽时引起的LES舒张。支配LES的交感传出神经起源于脊髓节段T6-T10。内脏节前纤维属于胆碱能神经元，它是以腹腔神经节为靶标激活去甲肾上腺素能节后神经元直接支配LES平滑肌，或与肠道运动神经元形成突触联系来完成对LES的支配。
溶血磷脂酸（Lysophosphatidic acid, LPA）是由血小板释放并存在于血清中的一种生物活性的脂质介体。它已经被确定是一种有效的并具有细胞增殖、存活和迁移、促进伤口愈合、血小板聚集、血管重塑、轴突回缩、分化抑制/逆转、膜去极化、形成粘着斑和应力纤维、血压调节和平滑肌收缩等多种生物学作用的磷脂信使。LPA具有其功能主要是通过结合其特定的G蛋白偶联受体（G protein-coupled receptors, GPCRs）而实现的。目前为止，一共有六个GPCRs被确定为特殊的LPA受体，它们是LPA1-6，其中LPA1-3被确定为内皮分化基因的GPCRS（Endothelial differentiation gene, EDG）亚家族的成员，因为它们彼此拥有很高的同源性。与此相反的，LPA4-6同LPA1-3同源性相差较大，它们属于非EDG家族成员。
大量研究表明，食管运动功能障碍性疾病如胡桃夹食管、贲门失弛缓症以及弥漫性食管痉挛等疾病中均与LES或/和食管肌层的功能异常相关。目前国内外的研究表明对于LES的调节机制主要涉及多种受体及其信号传导通路和多种神经递质都发挥着重要的作用。
本研究采用逆转录聚合酶链反应(RT-PCR)，实时定量聚合酶链反应（qPCR），蛋白印迹法（Western-blot），离体肌张力测定技术以及电场刺激（Electrical field stimulation, EFS）等方法对LPA受体在人LES中的表达及功能进行研究。探讨了LPA受体在人LES调节机制中的作用，为进一步研究LPA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，为治疗食管运动功能障碍性疾病提供依据。
第一部分溶血磷脂酸受体在人食管下括约肌的表达规律
目的：LPA受体是G蛋白偶联受体家族重要的受体之一。本实验采用逆转录聚合酶链反应（RT-PCR），实时定量聚合酶链反应（qPCR）和蛋白印迹法（Western-blot）研究人食管下括约肌中的LPA受体六种亚型在钩状纤维、套索纤维、食管环形肌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2012年1月到2012年7月因高位食管癌行食管大部切除术的患者总共15例，其中男性患者9例，女性患者6例，平均年龄约为64岁。在手术室收集新鲜的食管胃结合部标本，制备套索纤维、钩状纤维、胃底环形肌和食管环形肌肌条。提取各个组织总RNA，在确定它的纯度和它的完整性之后，分别应用6种溶血磷脂酸亚型的引物行RT-PCR，确定其mRNA在4种肌条的表达之后，在进行实时定量PCR确定其相对含量大小。提取各个组织总蛋白，将蛋白调整至相同的浓度，经电泳分离出LPA受体的各个亚型后转膜，随后分别应用LPA受体的各个亚型抗体进行孵育过夜，经Gel Pro软件分析各受体反应条带的光密度值（IOD）。
结果：紫外分光光度计测定显示，总RNA A260/A280比值约为1.8-2.0。六种LPA受体亚型的mRNA在各个肌条均有表达：分别是LPA1R、LPA2R、LPA3R、LPA4R、LPA5R和LPA6R。其扩增产物的长度与我们设计的长度是一致的。在同一种肌条中而不同的LPAR亚型的mRNA的表达水平的比较是有统计学差异的（F=61.034, P=0.000），它们的表达水平的强度依次为LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R。同一种LPA受体亚型的mRNA的表达水平在不同种肌条之间的比较没有统计学差异（F=0.201, P=0.895）。全部的LPA的受体亚型的总蛋白在四种肌条内也全部可以见其表达，分别是LPA1R、LPA2R、LPA3R、LPA4R、LPA5R和LPA6R。它们的分子量大小分别为39KD、40KD、42KD、42KD41KD和39KD。在同一种肌条内不同的LPAR亚型的总蛋白的表达的比较也是有统计学差异（F=1224.659, P=0.000），它们的蛋白表达水平的强弱和其mRNA的表达水平的结果是一致的。同一LPA受体亚型的蛋白表达水平在各个肌条间的比较是没有统计学差异的（F=0.039, P=0.990）。
结论：人LES中存在着六种LPA受体亚型，分别是LPA1R、LPA2R、LPA3R、 LPA4R、 LPA5R和LPA6R。它们的表达水平依次是LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R。它们可能在人LES的功能调节中发挥着重要作用。
第二部分溶血磷脂酸受体在人食管下括约肌调节机制中的功能研究
目的：研究非选择性的LPA受体的激动剂以及选择性的LPA受体的激动剂对人离体LES的钩状纤维（clasp）、套索纤维（sling）的作用，探讨LPA受体在人LES收缩和舒张的调节机制中所发挥的的作用。
方法：选取河北医科大学第四医院自2012年7月到2013年3月因高位食管癌行食管大部切除术的患者总共30例，其中男性患者19例，女性患者11例，平均年龄约为62岁。在手术室采集新鲜的食管胃结合部标本随后立即放入4℃的Krebs中，冲洗干净后将标本粘膜面向上固定在盛有Krebs液体的蜡盘内，保持含5％CO2和95％O2混合的气体持续通过。从标本的胃大弯侧切开，锐性剥离其贲门部和食管下段的粘膜层及粘膜下层，可见位于食管胃交界处的发白增厚的肌肉层即为食管下括约肌，肉眼识别出套索纤维与钩状纤维的位置。其套索纤维位于食管胃结合处的两端并呈斜行分布，而钩状纤维则位于食管胃结合处的中央并呈半环形。沿走形方向充分游离钩状纤维和套索纤维，并将其制备成（2~4）mm×（8~12）mm的肌条。分别用丝线将其肌条的两端扎紧，并置于含Krebs液10毫升保持37℃恒温的浴槽中，并持续通过其含有5％CO2和95％O2的气体。将肌条上端与JZ101型肌肉张力换能器固定在一起，应用Medlab信号采集器记录各个肌条的张力变化情况。缓慢轻柔的牵拉肌条使之张力达到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，每次牵拉的长度约为增加初始长度的25％左右，待其稳定后再次牵拉直至肌条长度被拉伸到初长度的200％作为其最适初长度。待肌条的最适初长度稳定大约40分钟以后，再以浓度累计方式分别向恒温浴槽中加入非选择性的LPA受体激动剂来激活LPA受体各个亚型。给药浓度为（10-9、10-8、10-7、10-6、10-5、10-4mol/L）。观察给药后肌条的张力变化的情况，待其张力稳定后可再增加一个浓度，每次加药前都要等前一个浓度达到其最大反应且稳定后加入下一浓度的药物。待肌条稳定10分钟后再行加药。据此建立累计给药的浓度-反应量效曲线。并计算出加药后反应的最大效应和其对应的浓度。在观察拮抗剂的效应时，发现其拮抗剂的浓度同激动剂诱导肌条产生最大效应的浓度是相同的。选择性的LPA受体激动剂同选择性的LPA拮抗剂的加药方法与之前相同。用肌条收缩或舒张百分比的均数±标准误（x±SE）来表示药物诱发的肌条的反应。
结果：
1非选择性LPA受体激动剂和拮抗剂对人LES的作用
非选择性LPA受体激动剂LPA在（10-6、10-5、10-4mol/L）的浓度能够诱导人LES的套索纤维、钩状纤维两种肌条产生收缩效应。应用非选择性LPA受体的阻滞剂（Tetradecyl-phosphonate）（10-5mol/L）可以完全抑制LPA（10-5mol/L）所诱导其套索纤维、钩状纤维产生的收缩效应。
2选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid对人LES的作用
选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid在（10-6、10-5、10-4mol/L）的浓度下能够诱导人LES中的套索纤维、钩状纤维这这两种肌条产生浓度依赖性的收缩效应。这两肌条间的收缩效应比较没有统计学差异（P＞0.05）。在10-5mol/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（25.3±1.1）％，套索纤维的最大收缩百分比为（23.8±0.9）％。这两者之间的比较无统计学差异（P＞0.05）。
3选择性的LPA3受体激动剂OMPT对人LES的作用
选择性的LPA3受体激动剂OMPT在（10-6、10-5、10-4mol/L）的浓度下能够诱导人LES中的套索纤维、钩状纤维这两种肌条产生浓度依赖性的收缩效应，这两肌条间的收缩效应比较没有统计学差异（P＞0.05）。在10-5mol/L时，这两肌条均可达到最大收缩百分比。钩状纤维的最大舒张百分比为（7.2±0.4）％，套索纤维的最大舒张百分比为（7.8±0.6）％，这两者的比较无统计学差异（P＞0.05）。
结论：
1非选择性LPA受体激动剂LPA能够诱导人的LES发生收缩效应。非选择性LPA受体的阻滞剂Tetradecyl-phosphonate能够完全抑制其非选择性激动剂LPA所诱导的收缩效应。这表明非选择性激动剂是通过LPA受体来实现其对食管下括约肌的调节作用的。
2选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid能够诱导人LES中的这两种肌条发生浓度依赖性的收缩效应。在10-5mol/L时，其达到了最大的收缩百分比。这两肌条间的收缩效应比较没有统计学差异。提示LPA1受体或LPA2受体，或LPA1和LPA2受体可能参与了食管下括约肌调节过程中的收缩反应。
3选择性的LPA3受体激动剂OMPT能够诱导人LES中的这两种肌条发生浓度依赖性的收缩效应，在10-5mol/L时，其达到了最大收缩百分比。这两肌条间的收缩效应比较没有统计学差异。提示LPA3受体可能也参与了食管下括约肌调节过程中的收缩反应。
第三部分溶血磷脂酸受体在电场刺激诱导的人食管下括约肌反应的作用研究
目的：研究选择性LPAR的拮抗剂在电场刺激下对人LES的套索纤维和钩状纤维的作用，探讨LPAR在人LES神经调节通路过程中的作用。
方法：选取河北医科大学第四医院自2013年3月到2013年12月因高位食管癌行食管大部切除术的患者总共20例，其中男性患者13例，女性患者7例，平均年龄约为59岁。套索纤维和钩状纤维的制备方法同前面所述。并将制备好的肌条置于含Krebs液10毫升并保持37℃恒温的浴槽中，并持续通过其含有5％CO2和95％O2的气体。将制备好的肌条的上端与肌肉张力换能器相连，同时下端同带有铂金电极的L形固定架固定在一起，应用Medlab信号采集器来记录各个肌条张力的变化情况。保证各个肌条在两个环形、平行的铂金电极的中间，它们之间的距离应该大于3mm，应用生理药理多用仪来进行电场刺激。应用上面所述的方法来调节肌条长度达到最适初长度。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。其频率从小到大行EFS，并计算出刺激后的最大效应（Emax）。待刺激停止后肌条恢复平衡以后，再分别依次向浴槽内加入其选择性的LPA1和LPA3受体的拮抗剂Ki16425（10-5mol/L）。在大约20分钟以后再分别行EFS，并进行加药前后EFS刺激对人LES出现反应的对比。用肌条的收缩或舒张百分比的均数±标准误（x±SE）来表示EFS刺激诱发的肌条的反应。
结果：
1EFS能够诱导人的食管下括约肌中的钩状纤维发生频率依赖性的舒张反应，其最大的舒张时的电刺激频率为64Hz，当每一次舒张反应结束以后，其肌条出现反跳性收缩，也是呈频率的依赖模式。EFS诱导钩状纤维的最大舒张百分比是17.8±0.7％。其套索纤维对EFS的反应表现呈频率依赖性的收缩反应，其最大的收缩时的电刺激频率为128Hz。EFS诱导套索纤维的最大收缩百分比是17.0±0.8％。
2选择性的LPA1和LPA3受体拮抗剂Ki16425在EFS引起的人食管下括约肌中的钩状和套索纤维频率依赖性舒张或收缩反应中的作用：选择性LPA1和LPA3受体拮抗剂Ki16425（10-5mol/L）对电场刺激诱发的人食管下括约肌中的钩状纤维频率依赖性舒张反应没有影响，对比其用药前后其舒张的效应没有统计学差异（P＞0.05）。选择性LPA1和LPA3受体拮抗剂Ki16425（10-5mol/L）对电场刺激诱发的人食管下括约肌中的套索纤维频率依赖性收缩反应没有影响，对比其用药前后收缩的效应没有统计学差异（P＞0.05）。
结论：
1电场刺激能够诱导人食管下括约肌中的钩状纤维发生频率依赖性的舒张反应。其最大舒张时的电刺激频率为64Hz。电场刺激能够诱导人食管下括约肌中的套索纤维发生频率依赖性收缩反应，其最大收缩时的电刺激频率为128Hz。
2应用选择性LPA1和LPA3受体拮抗剂Ki16425以后，对比其用药前后EFS所诱发人LES中的钩状纤维发生频率依赖性的舒张反应，其用药前后的舒张反应的变化没有统计学差异。提示在EFS所诱发的钩状纤维发生的频率依赖性的舒张反应中没有LPA1和LPA3受体的参与。
3应用选择性LPA1和LPA3受体拮抗剂Ki16425以后，对比其用药前后EFS所诱发人LES中的套索纤维发生频率依赖性的收缩反应，其用药前后的收缩反应的变化没有统计学差异。提示在EFS所诱发的套索纤维发生的频率依赖性的收缩反应中没有LPA1和LPA3受体的参与。
The esophagogastric junction (EGJ) is a complex valvular structure. The lower esophageal sphincter (LES) is one component of the EGJ that is conceptually a complex unit integrating both LES and diaphragmatic elements. The LES is a special thickened circular muscle layer about2-3cm in human and Liebermann-Meffer proposed firstly that the huanm lower esophageal sphincter consists of sling fibers at the greater curvature and clasp fibers at the lesser curvature in1979. It prevents reflux during nondeglutitive periods and facilitates flow during periods of opening.
The mechanism of contraction and relaxation of the LES is regulated by spontaneous myogenic factors, several hormones and neurotransmitters in the control of the central nervous system. The LES is innervated by vagal preganglionic and sympathetic postganglionic efferents. The vagal preganglionic fibers innervate the LES smooth muscle via ganglionic myenteric plexus neurons and provide both inhibitory and excitatory innervation to the LES. The inhibitory and excitatory preganglionic cell bodies are located within the caudal and rostal parts of the dorsal motor nucleus of the vagus, respectively. Typically cholinergic in nature, vagal preganglionic fibers exert their effects on the inhibitory myenteric plexus neurons via both nicotinic and muscarinic receptors. Excitatory and inhibitory neural activity influences LES resting tone by affecting the myogenic tone. Excitatory myenteric plexus neurons release Ach and substance P to increase LES pressure. The inhibitory myenteric plexus neurons decrease LES pressure with NO being the most dominant inhibitory neurotransmitter that is also involved in swallow-induced LES relaxation. The sympathetic efferents innervating the LES originate in spinal segments T6–T10. The splacnhnic preganglionic fibers are cholinergic and target the celiac ganglia to activate noradrenergic postganglionic neurons that either directly innervate LES smooth muscle or synapse onto the enteric motor neurons.
Lysophosphatidic acid (LPA) is a bioactive lipid mediator that is released by activated platelets and is constitutively present in serum. It has been identified to be a potent phospholipid messenger with a variety of biological actions, which include cell proliferation, survival and migration, wound healing, platelet aggregation, vascular remodeling, neurite retraction, differentiation inhibition/reversal, membrane depolarization, formation of focal adhesion and stress fibers, blood pressure regulation and smooth muscle contraction.
LPA exhibits its functions mainly through binding to its specific G protein-coupled receptors (GPCRs). Currently, there are six GPCRs identified as specific receptors for LPA that are referred to as LPA1-6. LPA1-3were identified as members of the endothelial differentiation gene (Edg) subfamily of GPCRS, as they share a high homology with each other. By contrast, LPA4-6belong to the non-Edg family.
It is demonstrated that esophageal motor disorders such as nutcracker esophagus, achalasia and diffuse esophageal spasm showed abnormalities of the lower esophageal sphincter or/and esophageal muscles. Recent studies have demonstrated that the regulatory mechanism of the LES involves various receptors, signal transduction pathways, and neurotransmitter, and play a role in the regulation of the LES at home and abroad.
Reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting, measurement of muscle tension in vitro, and electrical field stimulation (EFS) were used to identify expression and function of the LPA receptors in the human LES. The present study investigated the role that the LPA receptors play in modulating human LES function. So that we can demonstrate the regulatory mechanism of the LES much more properly, and provide theoretical bases for the clinical treatment of esophageal motility disorders.
PartⅠ Expression of LPA receptors in the human lower esophageal
sphincter
Objective: LPA receptor is a member of the G protein-coupled receptor family. In the present study, we identified the expression of mRNA and protein of LPA receptors in four muscle strips including sling fibers, clasp fibers, circular muscle strips of the esophagus and stomach by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting.
Methods: The muscle strips were selected from15patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from January2012to July2012for the study. There were9male and6female patients, with an average age of about64years. Fresh esophagogastric junction specimens were collected in the operating room, in which the sling fibers, clasp fibers and circular muscle strips of esophagus and stomach were separated. Total RNA was extracted. After the identification of its purity and integrity, reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers designed specifically to match the mRNA of LPA receptors. After mRNA expression in four kinds of muscle strips were determined, the relative expression level measued by real-time quantitative PCR. Total proteins were extracted from organizations, and adjusted to the same protein concentration. After electrophoretic separation of the various subtypes of LPA receptor they were transferred onto a polyvinylidene difluoride, respectively. At last the detection of the protein expression was operated using various LPA receptors’ polyclonal antibody. Gel Pro software was used to measured the integrated optical density (IOD) of each receptor bands.
Results: The value of A260/280of total RNA was between1.8and2.0after ultraviolet spectrophotometry. Transcripts for LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R were identified in the various muscle strips. The length of the amplification product was consistent with the expected size. Significant differences were demonstrated when comparing the expression of different LPA receptors’ mRNA in the same muscle strips (F=61.034, P= 0.000). The rank order of the extent of expression was LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R. However, there was no significant difference in mRNA expression of LPA receptors between the four muscle strips.(F=0.201, P=0.895). Protein expression of six LPA receptor subtypes were identified too, they were LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R, their molecular was39KD,40KD,42KD,42KD,41KD, and39KD, respectively. There was a significant difference in relative expression level for different LPA receptors in the same muscle strip (F=1224.659, P=0.000). The rank order of relative expression level was the same as the result of the RT-PCR. There was no significant difference in relative expression level between the four muscle strips.(F=0.039, P=0.990).
Conclusion: There are six kinds of LPA receptor subtypes in human LES, namely LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R. Their relative expression levels is LPA1R> LPA6R> LPA4R=LPA2R=LPA5R=LPA3R. They may play an important role in the regulation of human LES function.
Part Ⅱ The role of LPA receptors in modulating human lower esophageal sphincter
Objective: To identify the effects of selective and non-selective LPA receptors agonists on the sling fibers and clasp fibers of the human LES，and explore the regulatory mechanisms of LPA receptors in human LES contraction and relaxation.
Methods: The muscle strips were selected from30patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from July2012to March2013for the study. There were19male and11female patients, with an average age of about62years. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens were fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture. The specimen was cutted from the greater curvature of the stomach. Sharp dissection of its cardia mucosa and submucosa of the lower esophagus were performed and then the thickening of the muscle layer at the esophagogastric junction was found. The sling fibers could be identified in the gastric cardia, adjacent to the lesser curvature of the stomach. The clasp fibers could be identified adjacent to the lesser curvature of the stomach. The sling fibers and clasp fibers were dissociated and prepared into (2~4) mm×(8~12) mm muscle strips. Both ends of the muscle strips were fastened with silk, and placed in a10ml bath containing Krebs liquid, maintained a constant temperature of37℃and persisted through the gas containing5%CO2and95%O2. The upper of muscles and JZ101type muscle tension transducer were fastened together, Medlab signal acquisition applications recorded the changes in individual muscle tension. The muscle strips were pulled slowly and make the tension reaching200mg, this tension of the muscle strip is the initial length L0, Then the muscle strips were pulled slowly and repeatedly, each pulled about25%of the initial length, until the muscle strips were stretched to200%of the initial length which is as the optimum initial length. The optimal initial length of the muscle were stabilize about40minutes, then non-selective LPA receptor agonists were added into thermostatic bath to activate each LPA receptor subtypes, which was a cumulative manner from10-9to10-3mol/L. Each concentrations of the drug being added after the reaction of the previous concentration reached a maximum. The cumulative administration concentration-response dose-response curves were established due to the above results. And the maximum effect after dosing and its corresponding concentration were calculated. When observing the effects of antagonists, the concentration of the antagonist was found with a concentration of agonist-induced muscle maximal effect is the same. The administration of selective LPA receptors agonist and selective LPA receptors antagonist were in the same method. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error.
Results:
1Effect of non-selective LPA receptor agonist and antagonist on the human LES.
The non-selective dopamine receptor agonist LPA induced the contraction of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). The response induced by non-selective dopamine receptor agonist Tetradecyl-phosphonate at the concentration of10-5mol/L, was inhibited completely by non-selective dopamine receptor antagonist Tetradecyl-phosphonate (10-5mol/L).
2Effect of selective LPA1and LPA2receptor agonist on the human LES.
The selective LPA1and LPA2receptor agonist L-α-Lysophosphatidic acid induced a concentration-dependent contractile response of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/L. The maximum contraction percentage of clasp fibers was (25.3±1.1)％. The maximum contraction percentage of sling fibers was (23.8±0.9)％. There was no significant difference (P＞0.05).
3Effect of selective LPA3receptor agonist on the human LES.
The selective LPA3receptor agonist OMPT induced contraction of the human LES at the concentration of (10-6,10-5,10-4mol/L), which was in a concentration-dependent manner too. There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/l. The maximum contraction of clasp fibers was (7.2±0.4)％. The maximum relaxation of sling fibers was (7.8±0.6)％. There was no significant difference (P＞0.05).
Conclusions:
1The non-selective Lysophosphatidic acid receptor agonist can induce the contraction of the human LES. The reaction of the human LES can be inhibited completely by non-selective Lysophosphatidic acid receptor antagonist. This study indicates that LPA regulate the lower esophageal sphincter is through its receptor.
2The selective LPA1and LPA2receptor agonist induces a concentration-dependent contractile response. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA1or LPA2, or LPA1and LPA2receptor are involved in the contractile response of the human LES.
3The selective LPA3receptor agonist induces contraction of the human LES, which is in a concentration-dependent manner too. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA3receptor is involved in the contraction of the human LES.
PartⅢ The contribution of LPA receptors in the response of human lower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that LPA receptor subtypes play the role in the clasp fibers and sling fibers of the human lower esophageal sphincter (LES) under the electical field stimulation (EFS), and investigate the role of LPA receptor subtypes in vagal pathways that regulating human LES function.
Methods: The muscle strips were selected from20patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from March2013to December2013for the study. There were19male and11female patients, with an average age of about62years. The clasp fibers and sling fibers were prepared using the similar methods that were described previously. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens is fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture continued through. The upper of muscles and JZ101type muscle tension transducer were fastened together, while the lower end and an L-shaped bracket with platinum electrodes were fastened together. Medlab signal acquisition applications were used to record changes in individual muscle tension. All the muscles were ensured in the middle of the platinum electrode, which was more than3mm. Physiology and Pharmacology multi-purpose instrument was used for electrical field stimulation. The optimum initial length was ensured using the similar methods that were described previously. Electrical stimulation was conducted according to the frequency from small to big and the maximum effect after stimulation was calculated. Then the muscle strip was stimulated again after20min of administration of selective LPA1and LPA3receptor antagonist Ki16425at the concentration of10-5mol/L. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error.
Results:
1The EFS induced a frequency-dependent relaxation in clasp fibers of The LES. When the end of the relaxation response every time, there was a quick rebound contraction which was in the frequency-dependent manner too. The optimal frequency resulting in maximum relaxation percentage was64Hz. The maximum relaxation was (17.8±0.7)％.The EFS induced a frequency-dependent contraction in sling fibers. The optimal frequency resulting in maximum contraction percentage was128Hz. The maximum contraction was (17.0±0.8)％.
2Effect of the selective LPA1and LPA3receptor antagonist Ki16425on the clasp and sling fibers of the human LES under the EFS: The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced by the EFS (P＞0.05). The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent contraction in sling fibers of the human LES induced by the EFS (P＞0.05).
Conclusions:
1The EFS induce frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter. The optimal frequency resulting in maximum relaxation is64Hz. EFS induces frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter. The optimal frequency leading to maximum contraction is128Hz.
2The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that LPA1and LPA3receptors are not involved in the response of clasp fibers of the human lower esophageal sphincter induced by the EFS.
3The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that the LPA1and LPA3receptors are not involved in the response of sling fibers of the human lower esophageal sphincter induced by the EFS.
LES发生的收缩和舒张反应的调节机制是在中枢神经系统的支配下，由其自身肌源性因素、多种激素和神经递质参与而共同完成的。LES的收缩和舒张是由迷走神经节前纤维和交感神经节后纤维支配的。迷走神经节前纤维不仅可以通过肌间神经丛并且也可以通过抑制性神经通路和兴奋性神经通路来完成对LES的舒张和收缩功能的支配。其抑制性和兴奋性节前细胞分别位于迷走神经背侧运动核的尾部和前部。典型的是，胆碱能神经元是迷走神经节前纤维通过烟碱和毒蕈碱受体发挥其对肌间神经丛的抑制作用。兴奋性和抑制性神经元通路通过影响肌源性张力来影响LES张力。兴奋性肌间神经元通过释放乙酰胆碱和P物质增加LES张力。而抑制性肌间神经元是通过产生NO来减少LES的张力，NO是最主要的抑制性神经递质，在吞咽时引起的LES舒张。支配LES的交感传出神经起源于脊髓节段T6-T10。内脏节前纤维属于胆碱能神经元，它是以腹腔神经节为靶标激活去甲肾上腺素能节后神经元直接支配LES平滑肌，或与肠道运动神经元形成突触联系来完成对LES的支配。
溶血磷脂酸（Lysophosphatidic acid, LPA）是由血小板释放并存在于血清中的一种生物活性的脂质介体。它已经被确定是一种有效的并具有细胞增殖、存活和迁移、促进伤口愈合、血小板聚集、血管重塑、轴突回缩、分化抑制/逆转、膜去极化、形成粘着斑和应力纤维、血压调节和平滑肌收缩等多种生物学作用的磷脂信使。LPA具有其功能主要是通过结合其特定的G蛋白偶联受体（G protein-coupled receptors, GPCRs）而实现的。目前为止，一共有六个GPCRs被确定为特殊的LPA受体，它们是LPA1-6，其中LPA1-3被确定为内皮分化基因的GPCRS（Endothelial differentiation gene, EDG）亚家族的成员，因为它们彼此拥有很高的同源性。与此相反的，LPA4-6同LPA1-3同源性相差较大，它们属于非EDG家族成员。
大量研究表明，食管运动功能障碍性疾病如胡桃夹食管、贲门失弛缓症以及弥漫性食管痉挛等疾病中均与LES或/和食管肌层的功能异常相关。目前国内外的研究表明对于LES的调节机制主要涉及多种受体及其信号传导通路和多种神经递质都发挥着重要的作用。
本研究采用逆转录聚合酶链反应(RT-PCR)，实时定量聚合酶链反应（qPCR），蛋白印迹法（Western-blot），离体肌张力测定技术以及电场刺激（Electrical field stimulation, EFS）等方法对LPA受体在人LES中的表达及功能进行研究。探讨了LPA受体在人LES调节机制中的作用，为进一步研究LPA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，为治疗食管运动功能障碍性疾病提供依据。
第一部分溶血磷脂酸受体在人食管下括约肌的表达规律
目的：LPA受体是G蛋白偶联受体家族重要的受体之一。本实验采用逆转录聚合酶链反应（RT-PCR），实时定量聚合酶链反应（qPCR）和蛋白印迹法（Western-blot）研究人食管下括约肌中的LPA受体六种亚型在钩状纤维、套索纤维、食管环形肌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2012年1月到2012年7月因高位食管癌行食管大部切除术的患者总共15例，其中男性患者9例，女性患者6例，平均年龄约为64岁。在手术室收集新鲜的食管胃结合部标本，制备套索纤维、钩状纤维、胃底环形肌和食管环形肌肌条。提取各个组织总RNA，在确定它的纯度和它的完整性之后，分别应用6种溶血磷脂酸亚型的引物行RT-PCR，确定其mRNA在4种肌条的表达之后，在进行实时定量PCR确定其相对含量大小。提取各个组织总蛋白，将蛋白调整至相同的浓度，经电泳分离出LPA受体的各个亚型后转膜，随后分别应用LPA受体的各个亚型抗体进行孵育过夜，经Gel Pro软件分析各受体反应条带的光密度值（IOD）。
结果：紫外分光光度计测定显示，总RNA A260/A280比值约为1.8-2.0。六种LPA受体亚型的mRNA在各个肌条均有表达：分别是LPA1R、LPA2R、LPA3R、LPA4R、LPA5R和LPA6R。其扩增产物的长度与我们设计的长度是一致的。在同一种肌条中而不同的LPAR亚型的mRNA的表达水平的比较是有统计学差异的（F=61.034, P=0.000），它们的表达水平的强度依次为LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R。同一种LPA受体亚型的mRNA的表达水平在不同种肌条之间的比较没有统计学差异（F=0.201, P=0.895）。全部的LPA的受体亚型的总蛋白在四种肌条内也全部可以见其表达，分别是LPA1R、LPA2R、LPA3R、LPA4R、LPA5R和LPA6R。它们的分子量大小分别为39KD、40KD、42KD、42KD41KD和39KD。在同一种肌条内不同的LPAR亚型的总蛋白的表达的比较也是有统计学差异（F=1224.659, P=0.000），它们的蛋白表达水平的强弱和其mRNA的表达水平的结果是一致的。同一LPA受体亚型的蛋白表达水平在各个肌条间的比较是没有统计学差异的（F=0.039, P=0.990）。
结论：人LES中存在着六种LPA受体亚型，分别是LPA1R、LPA2R、LPA3R、 LPA4R、 LPA5R和LPA6R。它们的表达水平依次是LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R。它们可能在人LES的功能调节中发挥着重要作用。
第二部分溶血磷脂酸受体在人食管下括约肌调节机制中的功能研究
目的：研究非选择性的LPA受体的激动剂以及选择性的LPA受体的激动剂对人离体LES的钩状纤维（clasp）、套索纤维（sling）的作用，探讨LPA受体在人LES收缩和舒张的调节机制中所发挥的的作用。
方法：选取河北医科大学第四医院自2012年7月到2013年3月因高位食管癌行食管大部切除术的患者总共30例，其中男性患者19例，女性患者11例，平均年龄约为62岁。在手术室采集新鲜的食管胃结合部标本随后立即放入4℃的Krebs中，冲洗干净后将标本粘膜面向上固定在盛有Krebs液体的蜡盘内，保持含5％CO2和95％O2混合的气体持续通过。从标本的胃大弯侧切开，锐性剥离其贲门部和食管下段的粘膜层及粘膜下层，可见位于食管胃交界处的发白增厚的肌肉层即为食管下括约肌，肉眼识别出套索纤维与钩状纤维的位置。其套索纤维位于食管胃结合处的两端并呈斜行分布，而钩状纤维则位于食管胃结合处的中央并呈半环形。沿走形方向充分游离钩状纤维和套索纤维，并将其制备成（2~4）mm×（8~12）mm的肌条。分别用丝线将其肌条的两端扎紧，并置于含Krebs液10毫升保持37℃恒温的浴槽中，并持续通过其含有5％CO2和95％O2的气体。将肌条上端与JZ101型肌肉张力换能器固定在一起，应用Medlab信号采集器记录各个肌条的张力变化情况。缓慢轻柔的牵拉肌条使之张力达到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，每次牵拉的长度约为增加初始长度的25％左右，待其稳定后再次牵拉直至肌条长度被拉伸到初长度的200％作为其最适初长度。待肌条的最适初长度稳定大约40分钟以后，再以浓度累计方式分别向恒温浴槽中加入非选择性的LPA受体激动剂来激活LPA受体各个亚型。给药浓度为（10-9、10-8、10-7、10-6、10-5、10-4mol/L）。观察给药后肌条的张力变化的情况，待其张力稳定后可再增加一个浓度，每次加药前都要等前一个浓度达到其最大反应且稳定后加入下一浓度的药物。待肌条稳定10分钟后再行加药。据此建立累计给药的浓度-反应量效曲线。并计算出加药后反应的最大效应和其对应的浓度。在观察拮抗剂的效应时，发现其拮抗剂的浓度同激动剂诱导肌条产生最大效应的浓度是相同的。选择性的LPA受体激动剂同选择性的LPA拮抗剂的加药方法与之前相同。用肌条收缩或舒张百分比的均数±标准误（x±SE）来表示药物诱发的肌条的反应。
结果：
1非选择性LPA受体激动剂和拮抗剂对人LES的作用
非选择性LPA受体激动剂LPA在（10-6、10-5、10-4mol/L）的浓度能够诱导人LES的套索纤维、钩状纤维两种肌条产生收缩效应。应用非选择性LPA受体的阻滞剂（Tetradecyl-phosphonate）（10-5mol/L）可以完全抑制LPA（10-5mol/L）所诱导其套索纤维、钩状纤维产生的收缩效应。
2选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid对人LES的作用
选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid在（10-6、10-5、10-4mol/L）的浓度下能够诱导人LES中的套索纤维、钩状纤维这这两种肌条产生浓度依赖性的收缩效应。这两肌条间的收缩效应比较没有统计学差异（P＞0.05）。在10-5mol/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（25.3±1.1）％，套索纤维的最大收缩百分比为（23.8±0.9）％。这两者之间的比较无统计学差异（P＞0.05）。
3选择性的LPA3受体激动剂OMPT对人LES的作用
选择性的LPA3受体激动剂OMPT在（10-6、10-5、10-4mol/L）的浓度下能够诱导人LES中的套索纤维、钩状纤维这两种肌条产生浓度依赖性的收缩效应，这两肌条间的收缩效应比较没有统计学差异（P＞0.05）。在10-5mol/L时，这两肌条均可达到最大收缩百分比。钩状纤维的最大舒张百分比为（7.2±0.4）％，套索纤维的最大舒张百分比为（7.8±0.6）％，这两者的比较无统计学差异（P＞0.05）。
结论：
1非选择性LPA受体激动剂LPA能够诱导人的LES发生收缩效应。非选择性LPA受体的阻滞剂Tetradecyl-phosphonate能够完全抑制其非选择性激动剂LPA所诱导的收缩效应。这表明非选择性激动剂是通过LPA受体来实现其对食管下括约肌的调节作用的。
2选择性的LPA1和LPA2受体激动剂L-α-Lysophosphatidic acid能够诱导人LES中的这两种肌条发生浓度依赖性的收缩效应。在10-5mol/L时，其达到了最大的收缩百分比。这两肌条间的收缩效应比较没有统计学差异。提示LPA1受体或LPA2受体，或LPA1和LPA2受体可能参与了食管下括约肌调节过程中的收缩反应。
3选择性的LPA3受体激动剂OMPT能够诱导人LES中的这两种肌条发生浓度依赖性的收缩效应，在10-5mol/L时，其达到了最大收缩百分比。这两肌条间的收缩效应比较没有统计学差异。提示LPA3受体可能也参与了食管下括约肌调节过程中的收缩反应。
第三部分溶血磷脂酸受体在电场刺激诱导的人食管下括约肌反应的作用研究
目的：研究选择性LPAR的拮抗剂在电场刺激下对人LES的套索纤维和钩状纤维的作用，探讨LPAR在人LES神经调节通路过程中的作用。
方法：选取河北医科大学第四医院自2013年3月到2013年12月因高位食管癌行食管大部切除术的患者总共20例，其中男性患者13例，女性患者7例，平均年龄约为59岁。套索纤维和钩状纤维的制备方法同前面所述。并将制备好的肌条置于含Krebs液10毫升并保持37℃恒温的浴槽中，并持续通过其含有5％CO2和95％O2的气体。将制备好的肌条的上端与肌肉张力换能器相连，同时下端同带有铂金电极的L形固定架固定在一起，应用Medlab信号采集器来记录各个肌条张力的变化情况。保证各个肌条在两个环形、平行的铂金电极的中间，它们之间的距离应该大于3mm，应用生理药理多用仪来进行电场刺激。应用上面所述的方法来调节肌条长度达到最适初长度。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。其频率从小到大行EFS，并计算出刺激后的最大效应（Emax）。待刺激停止后肌条恢复平衡以后，再分别依次向浴槽内加入其选择性的LPA1和LPA3受体的拮抗剂Ki16425（10-5mol/L）。在大约20分钟以后再分别行EFS，并进行加药前后EFS刺激对人LES出现反应的对比。用肌条的收缩或舒张百分比的均数±标准误（x±SE）来表示EFS刺激诱发的肌条的反应。
结果：
1EFS能够诱导人的食管下括约肌中的钩状纤维发生频率依赖性的舒张反应，其最大的舒张时的电刺激频率为64Hz，当每一次舒张反应结束以后，其肌条出现反跳性收缩，也是呈频率的依赖模式。EFS诱导钩状纤维的最大舒张百分比是17.8±0.7％。其套索纤维对EFS的反应表现呈频率依赖性的收缩反应，其最大的收缩时的电刺激频率为128Hz。EFS诱导套索纤维的最大收缩百分比是17.0±0.8％。
2选择性的LPA1和LPA3受体拮抗剂Ki16425在EFS引起的人食管下括约肌中的钩状和套索纤维频率依赖性舒张或收缩反应中的作用：选择性LPA1和LPA3受体拮抗剂Ki16425（10-5mol/L）对电场刺激诱发的人食管下括约肌中的钩状纤维频率依赖性舒张反应没有影响，对比其用药前后其舒张的效应没有统计学差异（P＞0.05）。选择性LPA1和LPA3受体拮抗剂Ki16425（10-5mol/L）对电场刺激诱发的人食管下括约肌中的套索纤维频率依赖性收缩反应没有影响，对比其用药前后收缩的效应没有统计学差异（P＞0.05）。
结论：
1电场刺激能够诱导人食管下括约肌中的钩状纤维发生频率依赖性的舒张反应。其最大舒张时的电刺激频率为64Hz。电场刺激能够诱导人食管下括约肌中的套索纤维发生频率依赖性收缩反应，其最大收缩时的电刺激频率为128Hz。
2应用选择性LPA1和LPA3受体拮抗剂Ki16425以后，对比其用药前后EFS所诱发人LES中的钩状纤维发生频率依赖性的舒张反应，其用药前后的舒张反应的变化没有统计学差异。提示在EFS所诱发的钩状纤维发生的频率依赖性的舒张反应中没有LPA1和LPA3受体的参与。
3应用选择性LPA1和LPA3受体拮抗剂Ki16425以后，对比其用药前后EFS所诱发人LES中的套索纤维发生频率依赖性的收缩反应，其用药前后的收缩反应的变化没有统计学差异。提示在EFS所诱发的套索纤维发生的频率依赖性的收缩反应中没有LPA1和LPA3受体的参与。
The esophagogastric junction (EGJ) is a complex valvular structure. The lower esophageal sphincter (LES) is one component of the EGJ that is conceptually a complex unit integrating both LES and diaphragmatic elements. The LES is a special thickened circular muscle layer about2-3cm in human and Liebermann-Meffer proposed firstly that the huanm lower esophageal sphincter consists of sling fibers at the greater curvature and clasp fibers at the lesser curvature in1979. It prevents reflux during nondeglutitive periods and facilitates flow during periods of opening.
The mechanism of contraction and relaxation of the LES is regulated by spontaneous myogenic factors, several hormones and neurotransmitters in the control of the central nervous system. The LES is innervated by vagal preganglionic and sympathetic postganglionic efferents. The vagal preganglionic fibers innervate the LES smooth muscle via ganglionic myenteric plexus neurons and provide both inhibitory and excitatory innervation to the LES. The inhibitory and excitatory preganglionic cell bodies are located within the caudal and rostal parts of the dorsal motor nucleus of the vagus, respectively. Typically cholinergic in nature, vagal preganglionic fibers exert their effects on the inhibitory myenteric plexus neurons via both nicotinic and muscarinic receptors. Excitatory and inhibitory neural activity influences LES resting tone by affecting the myogenic tone. Excitatory myenteric plexus neurons release Ach and substance P to increase LES pressure. The inhibitory myenteric plexus neurons decrease LES pressure with NO being the most dominant inhibitory neurotransmitter that is also involved in swallow-induced LES relaxation. The sympathetic efferents innervating the LES originate in spinal segments T6–T10. The splacnhnic preganglionic fibers are cholinergic and target the celiac ganglia to activate noradrenergic postganglionic neurons that either directly innervate LES smooth muscle or synapse onto the enteric motor neurons.
Lysophosphatidic acid (LPA) is a bioactive lipid mediator that is released by activated platelets and is constitutively present in serum. It has been identified to be a potent phospholipid messenger with a variety of biological actions, which include cell proliferation, survival and migration, wound healing, platelet aggregation, vascular remodeling, neurite retraction, differentiation inhibition/reversal, membrane depolarization, formation of focal adhesion and stress fibers, blood pressure regulation and smooth muscle contraction.
LPA exhibits its functions mainly through binding to its specific G protein-coupled receptors (GPCRs). Currently, there are six GPCRs identified as specific receptors for LPA that are referred to as LPA1-6. LPA1-3were identified as members of the endothelial differentiation gene (Edg) subfamily of GPCRS, as they share a high homology with each other. By contrast, LPA4-6belong to the non-Edg family.
It is demonstrated that esophageal motor disorders such as nutcracker esophagus, achalasia and diffuse esophageal spasm showed abnormalities of the lower esophageal sphincter or/and esophageal muscles. Recent studies have demonstrated that the regulatory mechanism of the LES involves various receptors, signal transduction pathways, and neurotransmitter, and play a role in the regulation of the LES at home and abroad.
Reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting, measurement of muscle tension in vitro, and electrical field stimulation (EFS) were used to identify expression and function of the LPA receptors in the human LES. The present study investigated the role that the LPA receptors play in modulating human LES function. So that we can demonstrate the regulatory mechanism of the LES much more properly, and provide theoretical bases for the clinical treatment of esophageal motility disorders.
PartⅠ Expression of LPA receptors in the human lower esophageal
sphincter
Objective: LPA receptor is a member of the G protein-coupled receptor family. In the present study, we identified the expression of mRNA and protein of LPA receptors in four muscle strips including sling fibers, clasp fibers, circular muscle strips of the esophagus and stomach by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting.
Methods: The muscle strips were selected from15patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from January2012to July2012for the study. There were9male and6female patients, with an average age of about64years. Fresh esophagogastric junction specimens were collected in the operating room, in which the sling fibers, clasp fibers and circular muscle strips of esophagus and stomach were separated. Total RNA was extracted. After the identification of its purity and integrity, reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers designed specifically to match the mRNA of LPA receptors. After mRNA expression in four kinds of muscle strips were determined, the relative expression level measued by real-time quantitative PCR. Total proteins were extracted from organizations, and adjusted to the same protein concentration. After electrophoretic separation of the various subtypes of LPA receptor they were transferred onto a polyvinylidene difluoride, respectively. At last the detection of the protein expression was operated using various LPA receptors’ polyclonal antibody. Gel Pro software was used to measured the integrated optical density (IOD) of each receptor bands.
Results: The value of A260/280of total RNA was between1.8and2.0after ultraviolet spectrophotometry. Transcripts for LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R were identified in the various muscle strips. The length of the amplification product was consistent with the expected size. Significant differences were demonstrated when comparing the expression of different LPA receptors’ mRNA in the same muscle strips (F=61.034, P= 0.000). The rank order of the extent of expression was LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R. However, there was no significant difference in mRNA expression of LPA receptors between the four muscle strips.(F=0.201, P=0.895). Protein expression of six LPA receptor subtypes were identified too, they were LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R, their molecular was39KD,40KD,42KD,42KD,41KD, and39KD, respectively. There was a significant difference in relative expression level for different LPA receptors in the same muscle strip (F=1224.659, P=0.000). The rank order of relative expression level was the same as the result of the RT-PCR. There was no significant difference in relative expression level between the four muscle strips.(F=0.039, P=0.990).
Conclusion: There are six kinds of LPA receptor subtypes in human LES, namely LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R. Their relative expression levels is LPA1R> LPA6R> LPA4R=LPA2R=LPA5R=LPA3R. They may play an important role in the regulation of human LES function.
Part Ⅱ The role of LPA receptors in modulating human lower esophageal sphincter
Objective: To identify the effects of selective and non-selective LPA receptors agonists on the sling fibers and clasp fibers of the human LES，and explore the regulatory mechanisms of LPA receptors in human LES contraction and relaxation.
Methods: The muscle strips were selected from30patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from July2012to March2013for the study. There were19male and11female patients, with an average age of about62years. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens were fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture. The specimen was cutted from the greater curvature of the stomach. Sharp dissection of its cardia mucosa and submucosa of the lower esophagus were performed and then the thickening of the muscle layer at the esophagogastric junction was found. The sling fibers could be identified in the gastric cardia, adjacent to the lesser curvature of the stomach. The clasp fibers could be identified adjacent to the lesser curvature of the stomach. The sling fibers and clasp fibers were dissociated and prepared into (2~4) mm×(8~12) mm muscle strips. Both ends of the muscle strips were fastened with silk, and placed in a10ml bath containing Krebs liquid, maintained a constant temperature of37℃and persisted through the gas containing5%CO2and95%O2. The upper of muscles and JZ101type muscle tension transducer were fastened together, Medlab signal acquisition applications recorded the changes in individual muscle tension. The muscle strips were pulled slowly and make the tension reaching200mg, this tension of the muscle strip is the initial length L0, Then the muscle strips were pulled slowly and repeatedly, each pulled about25%of the initial length, until the muscle strips were stretched to200%of the initial length which is as the optimum initial length. The optimal initial length of the muscle were stabilize about40minutes, then non-selective LPA receptor agonists were added into thermostatic bath to activate each LPA receptor subtypes, which was a cumulative manner from10-9to10-3mol/L. Each concentrations of the drug being added after the reaction of the previous concentration reached a maximum. The cumulative administration concentration-response dose-response curves were established due to the above results. And the maximum effect after dosing and its corresponding concentration were calculated. When observing the effects of antagonists, the concentration of the antagonist was found with a concentration of agonist-induced muscle maximal effect is the same. The administration of selective LPA receptors agonist and selective LPA receptors antagonist were in the same method. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error.
Results:
1Effect of non-selective LPA receptor agonist and antagonist on the human LES.
The non-selective dopamine receptor agonist LPA induced the contraction of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). The response induced by non-selective dopamine receptor agonist Tetradecyl-phosphonate at the concentration of10-5mol/L, was inhibited completely by non-selective dopamine receptor antagonist Tetradecyl-phosphonate (10-5mol/L).
2Effect of selective LPA1and LPA2receptor agonist on the human LES.
The selective LPA1and LPA2receptor agonist L-α-Lysophosphatidic acid induced a concentration-dependent contractile response of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/L. The maximum contraction percentage of clasp fibers was (25.3±1.1)％. The maximum contraction percentage of sling fibers was (23.8±0.9)％. There was no significant difference (P＞0.05).
3Effect of selective LPA3receptor agonist on the human LES.
The selective LPA3receptor agonist OMPT induced contraction of the human LES at the concentration of (10-6,10-5,10-4mol/L), which was in a concentration-dependent manner too. There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/l. The maximum contraction of clasp fibers was (7.2±0.4)％. The maximum relaxation of sling fibers was (7.8±0.6)％. There was no significant difference (P＞0.05).
Conclusions:
1The non-selective Lysophosphatidic acid receptor agonist can induce the contraction of the human LES. The reaction of the human LES can be inhibited completely by non-selective Lysophosphatidic acid receptor antagonist. This study indicates that LPA regulate the lower esophageal sphincter is through its receptor.
2The selective LPA1and LPA2receptor agonist induces a concentration-dependent contractile response. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA1or LPA2, or LPA1and LPA2receptor are involved in the contractile response of the human LES.
3The selective LPA3receptor agonist induces contraction of the human LES, which is in a concentration-dependent manner too. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA3receptor is involved in the contraction of the human LES.
PartⅢ The contribution of LPA receptors in the response of human lower esophageal sphincter under the electical field stimulation
Objective: To identify the effect that LPA receptor subtypes play the role in the clasp fibers and sling fibers of the human lower esophageal sphincter (LES) under the electical field stimulation (EFS), and investigate the role of LPA receptor subtypes in vagal pathways that regulating human LES function.
Methods: The muscle strips were selected from20patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from March2013to December2013for the study. There were19male and11female patients, with an average age of about62years. The clasp fibers and sling fibers were prepared using the similar methods that were described previously. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens is fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture continued through. The upper of muscles and JZ101type muscle tension transducer were fastened together, while the lower end and an L-shaped bracket with platinum electrodes were fastened together. Medlab signal acquisition applications were used to record changes in individual muscle tension. All the muscles were ensured in the middle of the platinum electrode, which was more than3mm. Physiology and Pharmacology multi-purpose instrument was used for electrical field stimulation. The optimum initial length was ensured using the similar methods that were described previously. Electrical stimulation was conducted according to the frequency from small to big and the maximum effect after stimulation was calculated. Then the muscle strip was stimulated again after20min of administration of selective LPA1and LPA3receptor antagonist Ki16425at the concentration of10-5mol/L. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error.
Results:
1The EFS induced a frequency-dependent relaxation in clasp fibers of The LES. When the end of the relaxation response every time, there was a quick rebound contraction which was in the frequency-dependent manner too. The optimal frequency resulting in maximum relaxation percentage was64Hz. The maximum relaxation was (17.8±0.7)％.The EFS induced a frequency-dependent contraction in sling fibers. The optimal frequency resulting in maximum contraction percentage was128Hz. The maximum contraction was (17.0±0.8)％.
2Effect of the selective LPA1and LPA3receptor antagonist Ki16425on the clasp and sling fibers of the human LES under the EFS: The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced by the EFS (P＞0.05). The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent contraction in sling fibers of the human LES induced by the EFS (P＞0.05).
Conclusions:
1The EFS induce frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter. The optimal frequency resulting in maximum relaxation is64Hz. EFS induces frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter. The optimal frequency leading to maximum contraction is128Hz.
2The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that LPA1and LPA3receptors are not involved in the response of clasp fibers of the human lower esophageal sphincter induced by the EFS.
3The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that the LPA1and LPA3receptors are not involved in the response of sling fibers of the human lower esophageal sphincter induced by the EFS.
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1Eichholtz T, Jalink K, Fahrenfort I, et al. The bioactive phospholipidlysophosphatidic acid is released from activated platelets. Biochem J,1993,291:677~680
2Tigyi G, Hong L, Yakubu M, et al. Lysophosphatidic acid alterscerebrovascular reactivity in piglets. Am J Physiol,1995,268:H2048~H2055
3Jalink K, Hordijik PL and Moolenaar WH. Growth factor-like effects oflysophosphatidic acid, a novel lipid mediator. Biochim Biophys Acta,1994,1198:185~196
4Moolenaar WH. Lysophosphatidic acid signaling. Curr Opin Cell Biol,1995,7:203~210
5Gaits F, Fourcade O, Le Balle F, et al. Lysophosphatidic acid as aphospholipid mediator: pathways of synthesis. FEBS Lett,1997,410:54~58
6Nietgen G and Durieux ME. Intercellular signaling by lysophosphatidate.Cell Adhes Commun,1998,5:221~235
7Ishii I, Contos JJ, Fukushima N and Chun J. Functional comparisons of thelysophosphatidic acid receptors, LP(A1)/VZE-1/EDG-2, LP(A2)/EDG-4,and LP(A3)/EDG-7in neuronal cell lines using a retrovirus expressionsystem. Mol Pharmacol,2000,58:895~902
8Moolenaar WH, Van Meeteren LA and Giepmans BN. The ins and outs oflysophosphatidic acid signaling. Bioessays,2004,26:870~881
9Tokumura A, Fukuzawa K and Tsukatani H. Effects of synthetic andnatural lysophosphatidic acids in the arterial blood pressure of differentanimal species. Lipids,1978,13:572~574
10Salmon D. and Honeyman TW. Increased phosphatidate accumulationduring single contraction in isolated smooth muscle. Biochem. Soc.Transact,1979,7:631~640
11Tokumura A, Fukuzawa K, Yamada S, et al. Stimulatory effect oflysophosphatidic acids on uterine smooth muscles of non-pregnant rats.Arch Int Pharmacodyn Ther,1980,245:74~83
12Tokumura A, Fukuzawa K and Tsukatani H. Contractile actions oflysophosphatidic acids with a chemically-defined fatty acyl group onlongitudinal muscle from guineapig ileum. J Pharm Pharmacol,1982,34:514~516
13Tokumura A, Yube N, Fujimoto H and Tsukatani H. Lysophosphatidic acidsinduce contraction of rat isolated colon by two different mechanisms. JPharm Pharmacol,1991,43:774~778
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21An SZ, Bleu T, Hallmark OG and Goetzl EJ. Characterization of a novelsubtype of human G protein-coupled receptors for lysophosphatidic acid. JBiol Chem,1998,273:7906~7910
22Bandoh K, Aoki J, Hosono H, et al. Molecular cloning and characterizationof a novel human G protein-coupled receptor, EDG7, for lysophosphatidicacid. J Biol Chem,1999,274:27776~27785
23Noguchi K, Ishii S and Shimizu T. Identification of p2y9/GPR23as a novelG protein-coupled receptor for lysophosphatidic acid, structurally distantfrom the Edg family. J Biol Chem,2003,278:25600~25606
24Kotarsky K, Boketoft A, Bristulf J, et al. Lysophosphatidic acid binds toand activates GPR92, a G protein-coupled receptor highly expressed ingastrointestinal lymphocytes. J Pharmacol Exp Ther,2006,318:619~628
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1Eichholtz T, Jalink K, Fahrenfort I, et al. The bioactive phospholipidlysophosphatidic acid is released from activated platelets. Biochem J,1993,291:677~680
2Tigyi G, Hong L, Yakubu M, et al. Lysophosphatidic acid alterscerebrovascular reactivity in piglets. Am J Physiol,1995,268:H2048~H2055
3Jalink K, Hordijik PL and Moolenaar WH. Growth factor-like effects oflysophosphatidic acid, a novel lipid mediator. Biochim Biophys Acta,1994,1198:185~196
4Moolenaar WH. Lysophosphatidic acid signaling. Curr Opin Cell Biol,1995,7:203~210
5Gaits F, Fourcade O, Le Balle F, et al. Lysophosphatidic acid as aphospholipid mediator: pathways of synthesis. FEBS Lett,1997,410:54~58
6Nietgen G and Durieux ME. Intercellular signaling by lysophosphatidate.Cell Adhes Commun,1998,5:221~235
7Ishii I, Contos JJ, Fukushima N and Chun J. Functional comparisons of thelysophosphatidic acid receptors, LP(A1)/VZE-1/EDG-2, LP(A2)/EDG-4,and LP(A3)/EDG-7in neuronal cell lines using a retrovirus expressionsystem. Mol Pharmacol,2000,58:895~902
8Moolenaar WH, Van Meeteren LA and Giepmans BN. The ins and outs oflysophosphatidic acid signaling. Bioessays,2004,26:870~881
9Tokumura A, Fukuzawa K and Tsukatani H. Effects of synthetic andnatural lysophosphatidic acids in the arterial blood pressure of differentanimal species. Lipids,1978,13:572~574
10Salmon D. and Honeyman TW. Increased phosphatidate accumulationduring single contraction in isolated smooth muscle. Biochem. Soc.Transact,1979,7:631~640
11Tokumura A, Fukuzawa K, Yamada S, et al. Stimulatory effect oflysophosphatidic acids on uterine smooth muscles of non-pregnant rats.Arch Int Pharmacodyn Ther,1980,245:74~83
12Tokumura A, Fukuzawa K and Tsukatani H. Contractile actions oflysophosphatidic acids with a chemically-defined fatty acyl group onlongitudinal muscle from guineapig ileum. J Pharm Pharmacol,1982,34:514~516
13Tokumura A, Yube N, Fujimoto H and Tsukatani H. Lysophosphatidic acidsinduce contraction of rat isolated colon by two different mechanisms. JPharm Pharmacol,1991,43:774~778
14Toews ML, Ustinova EE and Schultz HD. Lysophosphatidic acid enhancescontractility of isolated airway smooth muscle. J Appl Physiol,1997,83:1216~1222
15Hecht JH, Weiner JA, Post SR and Chun J. Ventricular zone gene-1(vzg-1)encodes a lysophosphatidic acid receptor expressed in neurogenic regionsof the developing cerebral cortex. J Cell Biol,1996,135:1071~1083
16An SZ, Bleu T, Hallmark OG and Goetzl EJ. Characterization of a novelsubtype of human G protein-coupled receptors for lysophosphatidic acid. JBiol Chem,1998,273:7906~7910
17Bandoh K, Aoki J, Hosono H, et al. Molecular cloning and characterizationof a novel human G protein-coupled receptor, EDG7, for lysophosphatidicacid. J Biol Chem,1999,274:27776~27785
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