乙酰胆碱、蛙皮素和P物质对人类食管下括约肌张力调节的细胞内信号转导机制
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摘要
人食管下括约肌(lower esophageal sphincter, LES)是位于食管和胃交界部的一簇肌肉组织。它不仅可以保证食管内吞咽食物入胃畅通,而且能够阻止胃内容物向食管返流。当食管下括约肌的功能发生障碍时,就会造成食物通过障碍或胃食管返流疾病。对于人食管下括约肌的认识却经历了一个漫长的过程。直到1979年,Liebermann-Meffert等对食管胃连接部(esophago- gastric junction, EGJ)的平滑肌进行详细研究,提出人类食管下括约肌由内层大弯侧的套索纤维(sling fibers)和小弯侧半环形的钩状纤维(clasp fibers)构成,这两束肌肉共同维持LES的关闭状态,并形成了LES高压带(high- pressure zone, HPZ)。这一理论为进一步研究LES的生理、病理和药理学特性奠定了基础。在随后的研究中,学者们对不同动物LES的生理学特点和功能调节进行了较为深入的探讨,发现构成LES的套索纤维和钩状纤维在诸多方面存在着很大的差异性。
Sohn等在对动物LES的研究中发现,在猫的食管环形肌,由乙酰胆碱激动所引起的收缩由与PTX敏感的Gi3蛋白关联的M2型毒蕈碱受体介导,收缩依赖细胞外钙离子内流引发的磷脂酰胆碱特异性磷脂酶D(PC-PLD)的激活、DAG的产生和PKC依赖的信号转导途径。而在猫的食管下括约肌,乙酰胆碱则通过与PTX不敏感的Gq-G11蛋白相关联的M3受体起作用,其下游激活磷脂酰肌醇特异性磷脂酶C(PI-PLC),从而产生IP3并导致胞内钙的释放和钙调蛋白依赖的信号转导途径的激活。
Biancani等在对动物食管下括约肌单细胞的研究中,发现两条独立存在的导致收缩的信号转导通路。一条通路,由PI-PLC,IP3,依赖钙调蛋白的信号转导通路组成,通过最大有效剂量的乙酰胆碱激活。这条通路由M3受体与Gq-G11藕联,从而活化PLC,水解PIP2产生IP3和DAG。IP3导致胞内钙库释放Ca2+,产生钙-钙调蛋白复合体,肌球蛋白轻链磷酸化从而导致收缩。这条途径不依赖于PKC,这是因为钙调蛋白激动后抑制PKC的活性。在维持LES张力时或低于最大有效剂量的乙酰胆碱作用下,激活的则是一条PKC依赖转导通路,在这条通路中,PLC活性处于低水平,产生较少的IP3,从而使得胞内钙库释放较少的Ca2+,低水平的Ca2+不能激活钙调蛋白通路,然而PLC水解PIP2的另一产物DAG与低水平的IP3一起,则可以激活PKC依赖的转导通路。由此可见,钙离子的水平决定了介导食管下括约肌收缩的细胞内信号转导途径,高钙激活钙调蛋白通路,低钙激活PKC通路。
乙酰胆碱作为食管下括约肌张力调节的主要神经递质,在人食管下括约肌张力变化调节的过程中,是通过上述哪一条通路起作用,还是两条转导途径都有作用,目前还没有定论。乙酰胆碱引起肌细胞收缩的细胞内信号转导通路是激动剂-受体水平决定,还是因肌细胞部位的不同而不同,现在也不清楚。
因此,本研究选取广泛存在于中枢、外周及肠神经系统,对胃肠运动的调节与乙酰胆碱在受体水平不同的蛙皮素和P物质两种脑肠肽作比照,分析人食管下括约肌套索纤维,钩状纤维,食管环形肌和胃底环形肌细胞内信号转导途径,深入探讨构成LES的套索纤维和钩状纤维在细胞水平的异同,揭示两者在食管下括约肌张力变化调节中作用的内在机制。
第一部分乙酰胆碱、蛙皮素和P物质对人类食管下括约肌套索纤维和钩状纤维的调节作用
1乙酰胆碱对人类食管下括约肌套索纤维和钩状纤维的调节作用
目的:乙酰胆碱是食管下括约肌张力调节的主要神经递质,本文旨在通过研究乙酰胆碱对人食管下括约肌钩状纤维和套索纤维的作用规律,为研究受体后信号转导途径打下工作基础。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,得出各类肌细胞原长和不同终浓度的乙酰胆碱(mol/L: 10-11, 10-10, 10-9, 10-8, 10-7,10-6)作用收缩后的长度。通过软件SPSS 13.0进行统计学分析,得出浓度-反应关系曲线,求出各类肌细胞的最大收缩效应值(Emax)。并得出对应的激动剂浓度。所得数据以均数±标准差(x±s)表示,以P<0. 05为差异有统计学意义。
结果:在从10-11至10-6mol/L的浓度中,食管下括约肌的钩状纤维、套索纤维及食管环形肌、胃底环形肌四种类型的肌纤维,每种肌细胞均在乙酰胆碱10-9mol/L的浓度时取得最大收缩效应,并且10-9mol/L乙酰胆碱在钩状纤维、套索纤维及食管环形肌、胃底环形肌的收缩百分比分别为21.8±0.9%、23.1±1.9%、22.2±2.0%和21.9±1.2%。钩状纤维、套索纤维及食管环形肌、胃底环形肌组间差异无统计学意义(P>0.05)。
结论:人类LES的套索纤维和钩状纤维及食管环形肌、胃底环形肌的单个完整细胞对乙酰胆碱的刺激存在浓度依赖效应。在10-9mol/L乙酰胆碱的作用下取得最大收缩。
2蛙皮素和P物质对人类食管下括约肌套索纤维和钩状纤维的调节作用
目的:蛙皮素和P物质等脑肠肽广泛存在于中枢、外周及肠神经系统,对胃肠运动的调节与乙酰胆碱在受体水平不同。本文旨在通过研究蛙皮素和P物质对人食管下括约肌钩状纤维和套索纤维的作用规律,为研究受体后信号转导途径打下工作基础。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,得出各类肌细胞原长和不同终浓度的蛙皮素和P物质(mol/L: 10-11, 10-10, 10-9, 10-8, 10-7,10-6)作用收缩后的长度。通过软件SPSS 13.0进行统计学分析,得出浓度-反应关系曲线,求出各类肌细胞的最大收缩效应值(Emax)。并得出对应的激动剂浓度。所得数据以均数±标准差( x±s)表示,以P<0. 05为差异有统计学意义。
结果:在激动剂从10-11至10-6mol/L的浓度中,食管下括约肌的钩状纤维、套索纤维及食管环形肌、胃底环形肌四种类型的肌纤维,每种肌细胞均在蛙皮素和P物质10-7mol/L的浓度时取得最大收缩效应,并且10-7mol/L蛙皮素引起钩状纤维、套索纤维及食管环形肌、胃底环形肌的收缩百分比分别为23.2±1.1%、23.9±2.1%、23.9±1.2%和24.8±1.9%;10-7mol/L P物质分别为24.1±1.2%、25.8±1.1%、25.1±1.1%和24.3±2.1%,与乙酰胆碱比较,每种肌细胞三种激动剂所引起的最大收缩之间的差异无统计学意义(P>0.05)。而钩状纤维、套索纤维及食管环形肌、胃底环形肌之间的差异亦无统计学意义(P>0.05)。
结论:人类LES的套索纤维和钩状纤维及食管环形肌、胃底环形肌的单个完整细胞对蛙皮素和P物质的刺激存在浓度依赖效应。在10-7mol/L蛙皮素和P物质的作用下取得最大收缩。与乙酰胆碱比较,蛙皮素和P物质能够有效激动这四种肌细胞,取得与乙酰胆碱相近的最大收缩效应。
第二部分G蛋白在人类食管下括约肌套索纤维和钩状纤维的表达和功能
1 G蛋白在人食管下括约肌套索纤维和钩状纤维的表达
目的:乙酰胆碱、蛙皮素和P物质等激动剂使受体活化后,引起鸟苷酸结合蛋白(GTP-binding protein,G蛋白)的激活,作用于下游效应酶,从而影响胞内信使产生调控效应,介导食管下括约肌细胞的收缩。本文主要阐述G蛋白的四种亚型Gi3,Gq-G11,Go, Gs在人食管下括约肌套索纤维和钩状纤维的表达。
方法:收集2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者32例食管下括约肌的钩状纤维和套索纤维,以及食管环形肌和胃底环行肌四组标本。应用蛋白免疫印迹法(western blot)对比各组G蛋白亚型表达水平。结果以目的蛋白条带AU值与内参比较,凝胶成像软件分析结果。采用SPSS16.0统计软件。计量资料以平均值±标准差(x±s)表示,采用单因素方差分析,以P<0.05为差异有统计学意义。
结果: Western blot的结果显示,Gq/11和Gi3在人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌中均为宽条带,Go显示为细条带,Gs大体未见明显条带。凝胶成像软件分析结果:Gs蛋白条带吸光度值测不出,Gq/11的蛋白条带吸光度值(1组(套索纤维组):0.486±0.082;2组(钩状纤维组):0.492±0.109;3组(食管环形肌组):0.417±0.161;4组(胃底环形肌组):0.494±0.101),Go的蛋白条带吸光度值(1组:0.011±0.008;2组:0.016±0.012;3组:0.010±0.007;4组:0.009±0.008),Gi3的蛋白条带吸光度值(1组:0.567±0.101;2组:0.523±0.131;3组:0.590±0.097;4组:0.561±0.147)。组间比较差异均无统计学意义(P>0.05)。
结论:人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌中G蛋白的表达以Gq/11和Gi3为主,Go弱表达,Gs无表达。各G蛋白亚型表达在人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌的组间差异无统计学意义。
2 G蛋白在人食管下括约肌套索纤维和钩状纤维的功能
目的:最大有效剂量的乙酰胆碱、蛙皮素和P物质使受体活化后,引起鸟苷酸结合蛋白(GTP-binding protein,G蛋白)的激活,从而引起磷脂酶的活化,胞内信号分子水平的改变,食管下括约肌细胞的收缩。因此阐明介导食管下括约肌收缩过程中与各激动剂受体偶联的G蛋白的类型,对进一步研究受体后信号转导通路具有重要意义。
方法:收集2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者32例食管下括约肌的钩状纤维和套索纤维,以及食管环形肌和胃底环行肌四组标本。制备破膜平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,以乙酰胆碱、蛙皮素和P物质作为激动剂,选用一组特异性G蛋白抗体,对比不同亚型G蛋白抗体对各组肌细胞收缩的抑制情况,通过软件SPSS 16.0进行统计学分析,所得数据以均数±标准差(x±s)表示,采用单因素方差分析,以P<0.05为差异有统计学意义,分析受体偶联的G蛋白类型。
结果: 32例标本中,Gi3抗体对食管环形肌组和钩状纤维组的抑制作用明显强于Go,Gq/11和空白对照(P<0.001),说明在食管环形肌和钩状纤维,乙酰胆碱、蛙皮素和P物质引起的收缩依赖于G蛋白的Gi3亚型; Gq/11抗体对套索纤维和胃底环行肌组的抑制作用强于Go,Gi3和空白对照(P<0.001),说明在套索纤维和胃底环行肌,乙酰胆碱、蛙皮素和P物质引起的收缩依赖于G蛋白的Gq/11亚型。
结论:在以乙酰胆碱或蛙皮素、P物质作激动剂的试验中,食管环形肌和钩状纤维的收缩依赖于G蛋白的Gi3亚型。胃底环行肌和套索纤维的收缩依赖于G蛋白的Gq/11亚型。三种激动剂受体耦联的G蛋白类型具有肌细胞类型特异性和单一性的特点。Gi3和Gq/11分别在食管环形肌,钩状纤维和胃底环行肌,套索纤维离体细胞收缩过程中有重要作用。
第三部分磷脂酶在人类食管下括约肌套索纤维和钩状纤维收缩过程中的作用
目的:细胞外的激动剂和胞膜上的相应受体结合,激活相应的G蛋白后,需要细胞脂质池的水解,从而产生第二信使,将胞外的信号转化为细胞内信号,到达效应器,并最终形成细胞收缩动作。在这个过程当中,磷脂酶对于催化水解细胞脂质池起到了关键的作用,那么在研究的四种肌纤维中,激动剂的调节效应究竟是哪类磷脂酶活化并起到产生第二信使作用的,目前尚不清楚。本研究目的在于探讨磷脂酰肌醇特异的磷脂酶C,磷脂酰胆碱特异的磷脂酶C及磷脂酰胆碱特异的磷脂酶D在人食管下括约肌收缩过程中的作用规律。
方法:选取2008年3月至2005年10月在河北医科大学第四医院因高位食管中段癌行食管大部切除术患者5例,分离食管下括约肌套索纤维、钩状纤维和胃底环形肌、食管环形肌,制备单细胞悬液,皂苷作用制备破膜细胞,运用细胞动缘测量技术和药理学方法,观察磷脂酰肌醇特异的磷脂酶C的抑制剂U-73122(10-6M)、磷脂酰胆碱特异的磷脂酶C的抑制剂D609(10-4M)、磷脂酰胆碱特异的磷脂酶D的抑制剂普奈洛尔(10-4M)及D609、普奈洛尔合用四种方法处理对激动剂乙酰胆碱、P物质和蛙皮素收缩反应的抑制效应,用所占在无拮抗剂存在时细胞对激动剂的反应强度的百分比表示。以空白做对照。使用抑制剂U-73122,普奈洛尔和D609时,细胞在适当浓度的抑制剂中孵育5分钟后加入激动剂,抑制剂的使用浓度为其最大有效浓度。再应用细胞动缘学技术测定对四种肌细胞收缩的抑制情况。
结果: (1)PI-PLC的抑制剂U-73122作用时,对钩状纤维和食管环形肌细胞收缩的抑制程度较小,对套索纤维和胃底环形肌的抑制较明显。而钩状纤维,食管环形肌分别与套索纤维以及胃底环形肌比较,组间差异有统计学意义(p<0.05)。实验结果说明PI-PLC在乙酰胆碱、P物质和蛙皮素引起的套索纤维和胃底环形肌的收缩中起重要作用。(2)PC-PLC的抑制剂D609作用时,与PI-PLC的抑制剂U-73122相反,对套索纤维和胃底环形肌细胞收缩的抑制程度较小,对钩状纤维和食管环形肌的抑制较明显,而套索纤维,胃底环形肌分别与钩状纤维以及食管环形肌比较,组间差异有统计学意义(P<0.05)。实验结果说明PC-PLC在乙酰胆碱、P物质和蛙皮素引起的钩状纤维和食管环形肌的收缩中起重要作用。(3)PLD的抑制剂普奈洛尔作用时,对套索纤维和胃底环形肌细胞收缩的抑制程度较小,对钩状纤维和食管环形肌的抑制较明显,而套索纤维,胃底环形肌分别与钩状纤维以及食管环形肌比较,组间差异有统计学意义(P<0.05)。(4)D609和普奈洛尔合用时,对钩状纤维收缩的抑制与D609和普奈洛尔单独作用时比较(P=0.014,P=0.027),具有明显的叠加效应。
结论:乙酰胆碱,蛙皮素和P物质在套索纤维,胃底环形肌和钩状纤维,食管环形肌引起的收缩,活化的磷脂酶不同。抑制PI-PLC对套索纤维和胃底环形肌的收缩有抑制作用,抑制PC-PLC或PC-PLD对钩状纤维和食管环形肌的收缩有抑制作用,且两者共同作用具有叠加效应,几乎完全抑制钩状纤维和食管环形肌的收缩。
第四部分钙调蛋白信号通路和蛋白激酶C信号通路在人食管下括约肌中的作用
目的:PKC信号通路和钙调蛋白信号通路作为重要的细胞内信号转导途径,介导许多不同部位消化道平滑肌的收缩,是多种平滑肌细胞膜受体与相应的激动剂配体结合后启动,进而诱导肌细胞产生收缩效应的重要信号通路。本文旨在探讨蛋白激酶C(PKC)和钙调蛋白两条信号转导通路在人食管下括约肌钩状纤维和套索纤维收缩机制中的作用,并同时比较两种肌纤维与食管环形肌、胃底环形肌收缩机制的异同。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,分别应用PKC信号通路特异性阻断剂H7、无钙培养、钙调蛋白信号通路特异性阻断剂CGS9343B和以4mmol/L锶离子代替钙离子以阻断内源性钙离子的释放四种方法预处理后,加入激动剂,再测定对四种肌细胞收缩的抑制情况。
结果:无钙溶液可以阻断乙酰胆碱、蛙皮素和P物质诱导的钩状纤维和食管环形肌细胞的收缩,但对套索纤维和胃底环形肌收缩抑制不显著(P>0.05);以4mmol/L的锶离子代替胞外钙离子的方法,可以阻断乙酰胆碱、蛙皮素和P物质诱导的套索纤维和胃底环形肌细胞的收缩,但对钩状纤维和食管环形肌收缩抑制没有明显的影响(P>0.05);PKC信号通路特异性阻断剂H7明显抑制乙酰胆碱、蛙皮素和P物质诱导的钩状纤维和食管环形肌细胞的收缩,但对套索纤维和胃底环形肌收缩的抑制不明显(P>0.05);钙调蛋白信号通路特异性阻断剂CGS9343B可明显抑制乙酰胆碱、蛙皮素和P物质诱导的套索纤维和胃底环形肌细胞的收缩,但对钩状纤维和食管环形肌细胞的收缩没有显著影响(P>0.05)。
结论:钙调蛋白信号通路在介导套索纤维肌细胞收缩过程中起重要作用,而PKC信号通路在介导钩状纤维肌细胞收缩过程中起重要作用,两者分别需要细胞内源性钙离子的释放与细胞外钙离子的内流。
The lower esophageal sphincter (LES) in human beings lies between the esophagus and the stomach. It not only guarantees that food bolus in the esophagus pass into the stomach, but also prevent the gastric contents reflux into the esophagus. Thus, the structural or functional abnormality in the human LES may result in a number of disorders. Although the lower esophageal sphincter has great significance regarding the diagnosis and the treatment of esophageal benign disorders, the understandings of the lower esophageal sphincter in human beings have been a matter of speculation for many years. In 1979, Liebermann-Meffert et al described the arrangement of the smooth muscles at the esophagogastric junction (EGJ) in detail. They demonstrated that the musculature of the human LES consisted of sling fibers at the greater curvature and clasp fibers at the lesser curvature. This theory laid the foundation for further studies on the physiology, pathology and pharmacology of the LES. Over the ensuing years, many studies on the physiological feature and functional regulation of the LES in several kinds of animals were extensively conducted. These studies have suggested that the sling fibers and clasp fibers forming the LES varied significantly in many aspects.
Sohn et al. previously found that, Ach -induced contraction of cat esophageal circular muscle is mediated by a muscarinic M2 receptor linked to a PTX -sensitive Gi3 protein and depends on extracellular Ca2+-induced activation of PLD, production of DAG and activation of PKC. In contrast, in LES circular muscle, ACh acts through M3 muscarinic receptors linked to a PTX insensitive Gq-G11 protein, which activates PLC causing production of IP3, release of intracellular Ca2+ and activation of a calmodulin-dependent pathway.
Biancani et al. have found that two distinct contractile signal-transduction pathways are present in LES muscle cells. A PI-PLC, IP3, calmodulin-dependent pathway is activated by stimulation with a maximally effective dose of ACh. In this pathway, M3 muscarinic receptors link to Gq/11-type G proteins stimulate PLC, resulting in the formation of IP3 and DAG. IP3 causes the release of Ca2+ from intracellular stores, producing a calciumcalmodulin complex, myosin light chain phosphorylation, and contraction. This pathway is PKC independent, because maximal activation of calmodulin inhibits PKC activity. A distinct PKC-dependent pathway is activated by submaximal doses of ACh or during maintenance of LES tone. In this pathway, contraction is mediated by low levels of PLC activity, resulting in low levels of IP3, which cause the release of low levels of Ca2+ from intracellular stores. These low Ca2+ levels are insufficient to activate a calmodulindependent contraction, which requires micromolar Ca2+ concentrations. In addition, concurrent activity of a phosphatidylcholine -specific (PC-PLC) in the LES contributes to the production of DAG. Low levels of IP3 act synergistically with DAG to activate a PKC -dependent pathway. Thus the amount of Ca2+ available for contraction determines which pathway will be followed, with low Ca2+ levels activating a PKC-dependent pathway and high levels activating a calmodulin-dependent pathway.
As one of the most important neural transmitters, it is still not clear that which pathway or both in the LES tension regulation acetylcholine acts through. It is also not clear that the intracellular pathways activated by ACh may depend on the difference in receptors. Or alternatively, it is possible that distinct pathway may be a characteristic of the muscle types and that multiple receptors may activate only one pathway in one type of muscle cells and a different one in another.
In the present investigation, we examine bombesin and substance P along with acetylcholine, which are present in the enteric nervous system and utilize different receptors to cause contraction of gastrointestinal muscle. We examined the intracellular signal transduction pathways activated in human lower esophageal sphincter, both the sling fibers and the clasp fibers, as well as the circular layer muscle of the esophagus and gastric fundus. The differences of mechanisms in cellular level between the sling fibers and the clasp fibers in human lower esophageal sphincter tension regulation are also analyzed.
Part 1 Regulation of acetylcholine, bombesin and substance P on the sling fibers and clasp fibers of the human LES
1 Regulation of acetylcholine on the sling fibers and clasp fibers of the human LES
Objective: Acetylcholine is one of the most important neural transmitters regulating human lower esophageal sphincter tone. The research was conducted to investigate it’s regulation of the contraction of human sling fibers and clasp fibers forming LES, therefore to lay the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.
Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of acetylcholine (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 13.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-9mol/L for acetylcholine. The shortening percentages in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 21.8±0.9%、23.1±1.9%、22.2±2.0% and 21.9±1.2%. There were no significant differences in terms of the maximal response to acetylcholine between muscle types (P>0.05).
Conclusion: Acetylcholine causes contraction in a concentration- dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to acetylcholine occurs at a 10-9mol/L concentration.
2 Regulation of bombesin and substance P on the sling fibers and clasp fibers of the human LES
Objective: Bombesin and substance P which are present in the enteric nervous system differ from acetylcholine which binds to different receptor to cause contraction of gastrointestinal muscle. The research was conducted to investigate the effects of these agonists on the contraction of human sling fibers and clasp fibers forming LES, therefore to set up the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.
Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of bombesin and substance P (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 16.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-7mol/L for both bombesin and substance P. The shortening percentages caused by 10-7mol/L bombesin in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 23.2±1.1%、23.9±2.1%、23.9±1.2% and 24.8±1.9% ; The shortening percentages caused by 10-7mol/L substance P in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 24.1±1.2%、25.8±1.1%、25.1±1.1% and 24.3±2.1%. There were no significant differences in terms of the maximal response to both agonists and acetylcholine in any muscle type (P>0.05). And there were no significant differences between muscle types (P>0.05).
Conclusion: Bombesin and substance P cause contraction in a concentration-dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to bombesin and substance P occur at a 10-7mol/L concentration. The responses to bombesin and substance P are not different form the maximal response to acetylcholine.
Part 2 The expression and function of guanine nucleotide-binding protein on the human LES’sling fibers and clasp fibers
1 The expression of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LES
Objective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore the expression levels of four subtypes of guanine nucleotide-binding protein in the sling fibers and clasp fibers.
Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. The expressions of Gi3,Gq-G11,Go, Gs subtypes G proteins in these muscles were detected by Western blot. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant (by ANOVA).
Results: Gq/11 and Gi3 were detected as thick bands in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Go appeared as a thin band. Gs was barely visible.
Gel-analysis program showed the expression of Gq/11-subtype G protein in the sling fibers (0.486±0.082), in the clasp fibers (0.492±0.109), in the ESO (0.417±0.161) and in the gastric fundus (0.494±0.101); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Go-subtype G protein in the sling fibers (0.011±0.008), in the clasp fibers (0.016±0.012), in the ESO (0.010±0.007) and in the gastric fundus (0.009±0.008). Gs-subtype G protein was trace content in all four groups. The differences between groups were not statistically significant (P>0.05, by ANOVA).
Conclusion:These data suggest that Gq/11 and Gi3 are present in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Differences in each of four subtypes of G proteins in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus tissue are not apparent.
2 The function of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LES
Objective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore that which of four subtypes of guanine nucleotide-binding protein play an important role in the sling fibers and clasp fibers.
Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the four subtypes G protein antibodies before agonists were used. The original length and length changes of all kinds of muscle cells to different treatments were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. The maximal dose of acetylcholine, bombesin and substance P were used to induce contraction. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In the cells of the circular layers of the esophagus and the clasp fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gi3 antibodies (P < 0.001 by ANOVA), but not by Go, Gq/11 antibodies, which indicated that, in the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. While in the cells of the circular layers of the gastric fundus and the sling fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gq/11 antibodies (P < 0.001 by ANOVA) but not by Go, Gi3 antibodies, which indicated that, in the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11.
Conclusion:In the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. In the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11. Guanine nucleotide-binding proteins linked with acetylcholine, bombesin or substance P receptors are unique and muscle-type dependent.
Part 3 The role of phospholipases on the contraction of the human LES’sling fibers and clasp fibers
Objective:Activation of the phospholipases is responsible for hydrolysis of lipid pool and production of second messengers after guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P. Which kind of phospholipases is activated in human lower esophageal sphincter’s sling fibers and clasp fibers, and the circular layers of the esophagus and gastric fundus when stimulated by a maximally effective dose of Ach, bombesin or substance P is still not certain. This study was conducted to explore that the role of PC-PLC, PI-PLC, and PC-PLD on the human LES’sling fibers and clasp fibers.
Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the three phospholipases before agonists were used. When inhibitors (10-6M U-73122, 10-4M propranolol, 10-4M D609 and combination of propranolol and D609) were used, the cells were incubated in the antagonists for 5 min before addition of agonist, either acetylcholine, bombesin or substance P.
Results: (1) PI-PLC antagonist U-73122 (10-6 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the gastric fundus and the sling fibers, but not of the cells of the circular layers of the esophagus and the clasp fibers. The differences among four groups are significant (P<0.05). Which indicated that, PI-PLC played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the gastric fundus and the sling fibers. (2) Conversely, the PC- PLC inhibitor D609 (10-4 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). Which indicated that, PC-PLC plays an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers. (3) PC-PLD inhibitor propranolol (10-4M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). which indicated that, PC-PLD played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers.(4) In acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, the inhibitory effects of D609 and propranolol were additive. (P=0.014,P=0.027)by paired t test against D609 or propranolol alone.
Conclusion:PC-PLC, PI-PLC, and PC-PLD play different roles in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and gastric fundus, and the sling fibers and clasp fibers. The contraction of four muscle type cells in response to acetylcholine, bombesin or substance P is differentially inhibited by different phospholipase specific inhibitors. PI-PLC antagonist inhibits the contraction of the cells of the circular layers of the gastric fundus and the sling fibers; PC-PLC or PLD antagonists inhibits the contraction of the cells of the circular layers of the esophagus and the clasp fibers. And the inhibitory effects result in almost complete abolition of contraction.
PART 4 The role of calmodulin-dependent and PKC-dependent pathways on the contraction of the human LES’sling fibers and clasp fibers
Objective:To investigate the roles of two distinct intracellular signal transduction pathways: Protein Kinase C (PKC)-dependent and calmodulin- dependent, in the contractile mechanism of the clasp and sling fibers, the two parts of human lower esophageal sphincter, in comparison with circular muscle layers of gastric fundus and esophagus. Meanwhile the roles of extracellular and intracellular Ca2+ in this process were studied.
Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and October 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. When Protein Kinase C(PKC)-dependent pathway inhibitors H7, calmodulin-dependent pathway inhibitor CGS9343B, Ca2+-free medium or substitution of 4mmol/L Sr2+ for Ca2+ were used, the cells were incubated in appropriate solution for 5 min before addition of agonists. The length of cells was measured by video-based motion edge-detection system.
Results: Contraction of the cells of the clasp muscles and the esophagus was blocked by H7 and by incubation in Ca2+-free medium, but not by the CGS9343B and 4mmol/L Sr2+. By contrast, contraction of the cells from sling and gastric fundus was blocked only by CGS9343B and 4mmol/L Sr2+.
Conclusion: Contraction of the sling fiber and the circular muscle of the gastric fundus depends on release of intracellular calcium and activation of a calmodulin-dependent pathway. While contraction of the clasp fiber and the circular muscle of the esophagus depends on influx of extracellular calcium and activation of a PKC-dependent signal transduction pathway.
Sohn等在对动物LES的研究中发现,在猫的食管环形肌,由乙酰胆碱激动所引起的收缩由与PTX敏感的Gi3蛋白关联的M2型毒蕈碱受体介导,收缩依赖细胞外钙离子内流引发的磷脂酰胆碱特异性磷脂酶D(PC-PLD)的激活、DAG的产生和PKC依赖的信号转导途径。而在猫的食管下括约肌,乙酰胆碱则通过与PTX不敏感的Gq-G11蛋白相关联的M3受体起作用,其下游激活磷脂酰肌醇特异性磷脂酶C(PI-PLC),从而产生IP3并导致胞内钙的释放和钙调蛋白依赖的信号转导途径的激活。
Biancani等在对动物食管下括约肌单细胞的研究中,发现两条独立存在的导致收缩的信号转导通路。一条通路,由PI-PLC,IP3,依赖钙调蛋白的信号转导通路组成,通过最大有效剂量的乙酰胆碱激活。这条通路由M3受体与Gq-G11藕联,从而活化PLC,水解PIP2产生IP3和DAG。IP3导致胞内钙库释放Ca2+,产生钙-钙调蛋白复合体,肌球蛋白轻链磷酸化从而导致收缩。这条途径不依赖于PKC,这是因为钙调蛋白激动后抑制PKC的活性。在维持LES张力时或低于最大有效剂量的乙酰胆碱作用下,激活的则是一条PKC依赖转导通路,在这条通路中,PLC活性处于低水平,产生较少的IP3,从而使得胞内钙库释放较少的Ca2+,低水平的Ca2+不能激活钙调蛋白通路,然而PLC水解PIP2的另一产物DAG与低水平的IP3一起,则可以激活PKC依赖的转导通路。由此可见,钙离子的水平决定了介导食管下括约肌收缩的细胞内信号转导途径,高钙激活钙调蛋白通路,低钙激活PKC通路。
乙酰胆碱作为食管下括约肌张力调节的主要神经递质,在人食管下括约肌张力变化调节的过程中,是通过上述哪一条通路起作用,还是两条转导途径都有作用,目前还没有定论。乙酰胆碱引起肌细胞收缩的细胞内信号转导通路是激动剂-受体水平决定,还是因肌细胞部位的不同而不同,现在也不清楚。
因此,本研究选取广泛存在于中枢、外周及肠神经系统,对胃肠运动的调节与乙酰胆碱在受体水平不同的蛙皮素和P物质两种脑肠肽作比照,分析人食管下括约肌套索纤维,钩状纤维,食管环形肌和胃底环形肌细胞内信号转导途径,深入探讨构成LES的套索纤维和钩状纤维在细胞水平的异同,揭示两者在食管下括约肌张力变化调节中作用的内在机制。
第一部分乙酰胆碱、蛙皮素和P物质对人类食管下括约肌套索纤维和钩状纤维的调节作用
1乙酰胆碱对人类食管下括约肌套索纤维和钩状纤维的调节作用
目的:乙酰胆碱是食管下括约肌张力调节的主要神经递质,本文旨在通过研究乙酰胆碱对人食管下括约肌钩状纤维和套索纤维的作用规律,为研究受体后信号转导途径打下工作基础。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,得出各类肌细胞原长和不同终浓度的乙酰胆碱(mol/L: 10-11, 10-10, 10-9, 10-8, 10-7,10-6)作用收缩后的长度。通过软件SPSS 13.0进行统计学分析,得出浓度-反应关系曲线,求出各类肌细胞的最大收缩效应值(Emax)。并得出对应的激动剂浓度。所得数据以均数±标准差(x±s)表示,以P<0. 05为差异有统计学意义。
结果:在从10-11至10-6mol/L的浓度中,食管下括约肌的钩状纤维、套索纤维及食管环形肌、胃底环形肌四种类型的肌纤维,每种肌细胞均在乙酰胆碱10-9mol/L的浓度时取得最大收缩效应,并且10-9mol/L乙酰胆碱在钩状纤维、套索纤维及食管环形肌、胃底环形肌的收缩百分比分别为21.8±0.9%、23.1±1.9%、22.2±2.0%和21.9±1.2%。钩状纤维、套索纤维及食管环形肌、胃底环形肌组间差异无统计学意义(P>0.05)。
结论:人类LES的套索纤维和钩状纤维及食管环形肌、胃底环形肌的单个完整细胞对乙酰胆碱的刺激存在浓度依赖效应。在10-9mol/L乙酰胆碱的作用下取得最大收缩。
2蛙皮素和P物质对人类食管下括约肌套索纤维和钩状纤维的调节作用
目的:蛙皮素和P物质等脑肠肽广泛存在于中枢、外周及肠神经系统,对胃肠运动的调节与乙酰胆碱在受体水平不同。本文旨在通过研究蛙皮素和P物质对人食管下括约肌钩状纤维和套索纤维的作用规律,为研究受体后信号转导途径打下工作基础。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,得出各类肌细胞原长和不同终浓度的蛙皮素和P物质(mol/L: 10-11, 10-10, 10-9, 10-8, 10-7,10-6)作用收缩后的长度。通过软件SPSS 13.0进行统计学分析,得出浓度-反应关系曲线,求出各类肌细胞的最大收缩效应值(Emax)。并得出对应的激动剂浓度。所得数据以均数±标准差( x±s)表示,以P<0. 05为差异有统计学意义。
结果:在激动剂从10-11至10-6mol/L的浓度中,食管下括约肌的钩状纤维、套索纤维及食管环形肌、胃底环形肌四种类型的肌纤维,每种肌细胞均在蛙皮素和P物质10-7mol/L的浓度时取得最大收缩效应,并且10-7mol/L蛙皮素引起钩状纤维、套索纤维及食管环形肌、胃底环形肌的收缩百分比分别为23.2±1.1%、23.9±2.1%、23.9±1.2%和24.8±1.9%;10-7mol/L P物质分别为24.1±1.2%、25.8±1.1%、25.1±1.1%和24.3±2.1%,与乙酰胆碱比较,每种肌细胞三种激动剂所引起的最大收缩之间的差异无统计学意义(P>0.05)。而钩状纤维、套索纤维及食管环形肌、胃底环形肌之间的差异亦无统计学意义(P>0.05)。
结论:人类LES的套索纤维和钩状纤维及食管环形肌、胃底环形肌的单个完整细胞对蛙皮素和P物质的刺激存在浓度依赖效应。在10-7mol/L蛙皮素和P物质的作用下取得最大收缩。与乙酰胆碱比较,蛙皮素和P物质能够有效激动这四种肌细胞,取得与乙酰胆碱相近的最大收缩效应。
第二部分G蛋白在人类食管下括约肌套索纤维和钩状纤维的表达和功能
1 G蛋白在人食管下括约肌套索纤维和钩状纤维的表达
目的:乙酰胆碱、蛙皮素和P物质等激动剂使受体活化后,引起鸟苷酸结合蛋白(GTP-binding protein,G蛋白)的激活,作用于下游效应酶,从而影响胞内信使产生调控效应,介导食管下括约肌细胞的收缩。本文主要阐述G蛋白的四种亚型Gi3,Gq-G11,Go, Gs在人食管下括约肌套索纤维和钩状纤维的表达。
方法:收集2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者32例食管下括约肌的钩状纤维和套索纤维,以及食管环形肌和胃底环行肌四组标本。应用蛋白免疫印迹法(western blot)对比各组G蛋白亚型表达水平。结果以目的蛋白条带AU值与内参比较,凝胶成像软件分析结果。采用SPSS16.0统计软件。计量资料以平均值±标准差(x±s)表示,采用单因素方差分析,以P<0.05为差异有统计学意义。
结果: Western blot的结果显示,Gq/11和Gi3在人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌中均为宽条带,Go显示为细条带,Gs大体未见明显条带。凝胶成像软件分析结果:Gs蛋白条带吸光度值测不出,Gq/11的蛋白条带吸光度值(1组(套索纤维组):0.486±0.082;2组(钩状纤维组):0.492±0.109;3组(食管环形肌组):0.417±0.161;4组(胃底环形肌组):0.494±0.101),Go的蛋白条带吸光度值(1组:0.011±0.008;2组:0.016±0.012;3组:0.010±0.007;4组:0.009±0.008),Gi3的蛋白条带吸光度值(1组:0.567±0.101;2组:0.523±0.131;3组:0.590±0.097;4组:0.561±0.147)。组间比较差异均无统计学意义(P>0.05)。
结论:人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌中G蛋白的表达以Gq/11和Gi3为主,Go弱表达,Gs无表达。各G蛋白亚型表达在人食管下括约肌套索纤维钩状纤维以及食管环形肌,胃底环形肌的组间差异无统计学意义。
2 G蛋白在人食管下括约肌套索纤维和钩状纤维的功能
目的:最大有效剂量的乙酰胆碱、蛙皮素和P物质使受体活化后,引起鸟苷酸结合蛋白(GTP-binding protein,G蛋白)的激活,从而引起磷脂酶的活化,胞内信号分子水平的改变,食管下括约肌细胞的收缩。因此阐明介导食管下括约肌收缩过程中与各激动剂受体偶联的G蛋白的类型,对进一步研究受体后信号转导通路具有重要意义。
方法:收集2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者32例食管下括约肌的钩状纤维和套索纤维,以及食管环形肌和胃底环行肌四组标本。制备破膜平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,以乙酰胆碱、蛙皮素和P物质作为激动剂,选用一组特异性G蛋白抗体,对比不同亚型G蛋白抗体对各组肌细胞收缩的抑制情况,通过软件SPSS 16.0进行统计学分析,所得数据以均数±标准差(x±s)表示,采用单因素方差分析,以P<0.05为差异有统计学意义,分析受体偶联的G蛋白类型。
结果: 32例标本中,Gi3抗体对食管环形肌组和钩状纤维组的抑制作用明显强于Go,Gq/11和空白对照(P<0.001),说明在食管环形肌和钩状纤维,乙酰胆碱、蛙皮素和P物质引起的收缩依赖于G蛋白的Gi3亚型; Gq/11抗体对套索纤维和胃底环行肌组的抑制作用强于Go,Gi3和空白对照(P<0.001),说明在套索纤维和胃底环行肌,乙酰胆碱、蛙皮素和P物质引起的收缩依赖于G蛋白的Gq/11亚型。
结论:在以乙酰胆碱或蛙皮素、P物质作激动剂的试验中,食管环形肌和钩状纤维的收缩依赖于G蛋白的Gi3亚型。胃底环行肌和套索纤维的收缩依赖于G蛋白的Gq/11亚型。三种激动剂受体耦联的G蛋白类型具有肌细胞类型特异性和单一性的特点。Gi3和Gq/11分别在食管环形肌,钩状纤维和胃底环行肌,套索纤维离体细胞收缩过程中有重要作用。
第三部分磷脂酶在人类食管下括约肌套索纤维和钩状纤维收缩过程中的作用
目的:细胞外的激动剂和胞膜上的相应受体结合,激活相应的G蛋白后,需要细胞脂质池的水解,从而产生第二信使,将胞外的信号转化为细胞内信号,到达效应器,并最终形成细胞收缩动作。在这个过程当中,磷脂酶对于催化水解细胞脂质池起到了关键的作用,那么在研究的四种肌纤维中,激动剂的调节效应究竟是哪类磷脂酶活化并起到产生第二信使作用的,目前尚不清楚。本研究目的在于探讨磷脂酰肌醇特异的磷脂酶C,磷脂酰胆碱特异的磷脂酶C及磷脂酰胆碱特异的磷脂酶D在人食管下括约肌收缩过程中的作用规律。
方法:选取2008年3月至2005年10月在河北医科大学第四医院因高位食管中段癌行食管大部切除术患者5例,分离食管下括约肌套索纤维、钩状纤维和胃底环形肌、食管环形肌,制备单细胞悬液,皂苷作用制备破膜细胞,运用细胞动缘测量技术和药理学方法,观察磷脂酰肌醇特异的磷脂酶C的抑制剂U-73122(10-6M)、磷脂酰胆碱特异的磷脂酶C的抑制剂D609(10-4M)、磷脂酰胆碱特异的磷脂酶D的抑制剂普奈洛尔(10-4M)及D609、普奈洛尔合用四种方法处理对激动剂乙酰胆碱、P物质和蛙皮素收缩反应的抑制效应,用所占在无拮抗剂存在时细胞对激动剂的反应强度的百分比表示。以空白做对照。使用抑制剂U-73122,普奈洛尔和D609时,细胞在适当浓度的抑制剂中孵育5分钟后加入激动剂,抑制剂的使用浓度为其最大有效浓度。再应用细胞动缘学技术测定对四种肌细胞收缩的抑制情况。
结果: (1)PI-PLC的抑制剂U-73122作用时,对钩状纤维和食管环形肌细胞收缩的抑制程度较小,对套索纤维和胃底环形肌的抑制较明显。而钩状纤维,食管环形肌分别与套索纤维以及胃底环形肌比较,组间差异有统计学意义(p<0.05)。实验结果说明PI-PLC在乙酰胆碱、P物质和蛙皮素引起的套索纤维和胃底环形肌的收缩中起重要作用。(2)PC-PLC的抑制剂D609作用时,与PI-PLC的抑制剂U-73122相反,对套索纤维和胃底环形肌细胞收缩的抑制程度较小,对钩状纤维和食管环形肌的抑制较明显,而套索纤维,胃底环形肌分别与钩状纤维以及食管环形肌比较,组间差异有统计学意义(P<0.05)。实验结果说明PC-PLC在乙酰胆碱、P物质和蛙皮素引起的钩状纤维和食管环形肌的收缩中起重要作用。(3)PLD的抑制剂普奈洛尔作用时,对套索纤维和胃底环形肌细胞收缩的抑制程度较小,对钩状纤维和食管环形肌的抑制较明显,而套索纤维,胃底环形肌分别与钩状纤维以及食管环形肌比较,组间差异有统计学意义(P<0.05)。(4)D609和普奈洛尔合用时,对钩状纤维收缩的抑制与D609和普奈洛尔单独作用时比较(P=0.014,P=0.027),具有明显的叠加效应。
结论:乙酰胆碱,蛙皮素和P物质在套索纤维,胃底环形肌和钩状纤维,食管环形肌引起的收缩,活化的磷脂酶不同。抑制PI-PLC对套索纤维和胃底环形肌的收缩有抑制作用,抑制PC-PLC或PC-PLD对钩状纤维和食管环形肌的收缩有抑制作用,且两者共同作用具有叠加效应,几乎完全抑制钩状纤维和食管环形肌的收缩。
第四部分钙调蛋白信号通路和蛋白激酶C信号通路在人食管下括约肌中的作用
目的:PKC信号通路和钙调蛋白信号通路作为重要的细胞内信号转导途径,介导许多不同部位消化道平滑肌的收缩,是多种平滑肌细胞膜受体与相应的激动剂配体结合后启动,进而诱导肌细胞产生收缩效应的重要信号通路。本文旨在探讨蛋白激酶C(PKC)和钙调蛋白两条信号转导通路在人食管下括约肌钩状纤维和套索纤维收缩机制中的作用,并同时比较两种肌纤维与食管环形肌、胃底环形肌收缩机制的异同。
方法:选取2008年3月至2009年10月在河北医科大学第四医院因食管中段癌行食管大部切除术患者5例,采集套索纤维、钩状纤维、食管体部和胃底部的环行肌组织。制备平滑肌单细胞悬液,利用细胞动缘测量技术和药理学方法,分别应用PKC信号通路特异性阻断剂H7、无钙培养、钙调蛋白信号通路特异性阻断剂CGS9343B和以4mmol/L锶离子代替钙离子以阻断内源性钙离子的释放四种方法预处理后,加入激动剂,再测定对四种肌细胞收缩的抑制情况。
结果:无钙溶液可以阻断乙酰胆碱、蛙皮素和P物质诱导的钩状纤维和食管环形肌细胞的收缩,但对套索纤维和胃底环形肌收缩抑制不显著(P>0.05);以4mmol/L的锶离子代替胞外钙离子的方法,可以阻断乙酰胆碱、蛙皮素和P物质诱导的套索纤维和胃底环形肌细胞的收缩,但对钩状纤维和食管环形肌收缩抑制没有明显的影响(P>0.05);PKC信号通路特异性阻断剂H7明显抑制乙酰胆碱、蛙皮素和P物质诱导的钩状纤维和食管环形肌细胞的收缩,但对套索纤维和胃底环形肌收缩的抑制不明显(P>0.05);钙调蛋白信号通路特异性阻断剂CGS9343B可明显抑制乙酰胆碱、蛙皮素和P物质诱导的套索纤维和胃底环形肌细胞的收缩,但对钩状纤维和食管环形肌细胞的收缩没有显著影响(P>0.05)。
结论:钙调蛋白信号通路在介导套索纤维肌细胞收缩过程中起重要作用,而PKC信号通路在介导钩状纤维肌细胞收缩过程中起重要作用,两者分别需要细胞内源性钙离子的释放与细胞外钙离子的内流。
The lower esophageal sphincter (LES) in human beings lies between the esophagus and the stomach. It not only guarantees that food bolus in the esophagus pass into the stomach, but also prevent the gastric contents reflux into the esophagus. Thus, the structural or functional abnormality in the human LES may result in a number of disorders. Although the lower esophageal sphincter has great significance regarding the diagnosis and the treatment of esophageal benign disorders, the understandings of the lower esophageal sphincter in human beings have been a matter of speculation for many years. In 1979, Liebermann-Meffert et al described the arrangement of the smooth muscles at the esophagogastric junction (EGJ) in detail. They demonstrated that the musculature of the human LES consisted of sling fibers at the greater curvature and clasp fibers at the lesser curvature. This theory laid the foundation for further studies on the physiology, pathology and pharmacology of the LES. Over the ensuing years, many studies on the physiological feature and functional regulation of the LES in several kinds of animals were extensively conducted. These studies have suggested that the sling fibers and clasp fibers forming the LES varied significantly in many aspects.
Sohn et al. previously found that, Ach -induced contraction of cat esophageal circular muscle is mediated by a muscarinic M2 receptor linked to a PTX -sensitive Gi3 protein and depends on extracellular Ca2+-induced activation of PLD, production of DAG and activation of PKC. In contrast, in LES circular muscle, ACh acts through M3 muscarinic receptors linked to a PTX insensitive Gq-G11 protein, which activates PLC causing production of IP3, release of intracellular Ca2+ and activation of a calmodulin-dependent pathway.
Biancani et al. have found that two distinct contractile signal-transduction pathways are present in LES muscle cells. A PI-PLC, IP3, calmodulin-dependent pathway is activated by stimulation with a maximally effective dose of ACh. In this pathway, M3 muscarinic receptors link to Gq/11-type G proteins stimulate PLC, resulting in the formation of IP3 and DAG. IP3 causes the release of Ca2+ from intracellular stores, producing a calciumcalmodulin complex, myosin light chain phosphorylation, and contraction. This pathway is PKC independent, because maximal activation of calmodulin inhibits PKC activity. A distinct PKC-dependent pathway is activated by submaximal doses of ACh or during maintenance of LES tone. In this pathway, contraction is mediated by low levels of PLC activity, resulting in low levels of IP3, which cause the release of low levels of Ca2+ from intracellular stores. These low Ca2+ levels are insufficient to activate a calmodulindependent contraction, which requires micromolar Ca2+ concentrations. In addition, concurrent activity of a phosphatidylcholine -specific (PC-PLC) in the LES contributes to the production of DAG. Low levels of IP3 act synergistically with DAG to activate a PKC -dependent pathway. Thus the amount of Ca2+ available for contraction determines which pathway will be followed, with low Ca2+ levels activating a PKC-dependent pathway and high levels activating a calmodulin-dependent pathway.
As one of the most important neural transmitters, it is still not clear that which pathway or both in the LES tension regulation acetylcholine acts through. It is also not clear that the intracellular pathways activated by ACh may depend on the difference in receptors. Or alternatively, it is possible that distinct pathway may be a characteristic of the muscle types and that multiple receptors may activate only one pathway in one type of muscle cells and a different one in another.
In the present investigation, we examine bombesin and substance P along with acetylcholine, which are present in the enteric nervous system and utilize different receptors to cause contraction of gastrointestinal muscle. We examined the intracellular signal transduction pathways activated in human lower esophageal sphincter, both the sling fibers and the clasp fibers, as well as the circular layer muscle of the esophagus and gastric fundus. The differences of mechanisms in cellular level between the sling fibers and the clasp fibers in human lower esophageal sphincter tension regulation are also analyzed.
Part 1 Regulation of acetylcholine, bombesin and substance P on the sling fibers and clasp fibers of the human LES
1 Regulation of acetylcholine on the sling fibers and clasp fibers of the human LES
Objective: Acetylcholine is one of the most important neural transmitters regulating human lower esophageal sphincter tone. The research was conducted to investigate it’s regulation of the contraction of human sling fibers and clasp fibers forming LES, therefore to lay the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.
Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of acetylcholine (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 13.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-9mol/L for acetylcholine. The shortening percentages in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 21.8±0.9%、23.1±1.9%、22.2±2.0% and 21.9±1.2%. There were no significant differences in terms of the maximal response to acetylcholine between muscle types (P>0.05).
Conclusion: Acetylcholine causes contraction in a concentration- dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to acetylcholine occurs at a 10-9mol/L concentration.
2 Regulation of bombesin and substance P on the sling fibers and clasp fibers of the human LES
Objective: Bombesin and substance P which are present in the enteric nervous system differ from acetylcholine which binds to different receptor to cause contraction of gastrointestinal muscle. The research was conducted to investigate the effects of these agonists on the contraction of human sling fibers and clasp fibers forming LES, therefore to set up the foundation for investigating intracellular signal transduction pathways in human lower esophageal sphincter.
Methods: Smooth muscles of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus were obtained from 5 patients who underwent subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009. All patients gave informed consent to the study. Suspension of smooth muscle cells from the sling fibers and clasp fibers were prepared. The original length and length changes of all kinds of muscle cells to different concentrations of bombesin and substance P (mol/L: 10-11, 10-10, 10-9, 10-8, 10-7, 10-6) were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. Concentration-response curves of the cells were created from the obtained data by using SPSS 16.0 software, and from which the maximal responses and the corresponding concentrations obtained. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In smooth muscle cells of the sling and clasp fibers, the circular layer of the esophagus and gastric fundus, maximal contractile effect could be obtained at the concentration of 10-7mol/L for both bombesin and substance P. The shortening percentages caused by 10-7mol/L bombesin in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 23.2±1.1%、23.9±2.1%、23.9±1.2% and 24.8±1.9% ; The shortening percentages caused by 10-7mol/L substance P in the clasp fibers, sling fibers, the circular layer of the esophagus and gastric fundus were: 24.1±1.2%、25.8±1.1%、25.1±1.1% and 24.3±2.1%. There were no significant differences in terms of the maximal response to both agonists and acetylcholine in any muscle type (P>0.05). And there were no significant differences between muscle types (P>0.05).
Conclusion: Bombesin and substance P cause contraction in a concentration-dependent manner in the single intact cells of human LES, both sling fibers and clasp fibers, the circular layer of the esophagus and gastric fundus. The maximal response to bombesin and substance P occur at a 10-7mol/L concentration. The responses to bombesin and substance P are not different form the maximal response to acetylcholine.
Part 2 The expression and function of guanine nucleotide-binding protein on the human LES’sling fibers and clasp fibers
1 The expression of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LES
Objective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore the expression levels of four subtypes of guanine nucleotide-binding protein in the sling fibers and clasp fibers.
Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. The expressions of Gi3,Gq-G11,Go, Gs subtypes G proteins in these muscles were detected by Western blot. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant (by ANOVA).
Results: Gq/11 and Gi3 were detected as thick bands in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Go appeared as a thin band. Gs was barely visible.
Gel-analysis program showed the expression of Gq/11-subtype G protein in the sling fibers (0.486±0.082), in the clasp fibers (0.492±0.109), in the ESO (0.417±0.161) and in the gastric fundus (0.494±0.101); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Gi3-subtype G protein in the sling fibers (0.567±0.101), in the clasp fibers (0.523±0.131), in the ESO (0.590±0.097) and in the gastric fundus (0.561±0.147); the expression of Go-subtype G protein in the sling fibers (0.011±0.008), in the clasp fibers (0.016±0.012), in the ESO (0.010±0.007) and in the gastric fundus (0.009±0.008). Gs-subtype G protein was trace content in all four groups. The differences between groups were not statistically significant (P>0.05, by ANOVA).
Conclusion:These data suggest that Gq/11 and Gi3 are present in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus. Differences in each of four subtypes of G proteins in the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus tissue are not apparent.
2 The function of guanine nucleotide-binding protein on the sling fibers and clasp fibers of the human LES
Objective : Guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P, resulting in the activation of phospholipase, changes of intracellular messenger levels and contraction of lower esophageal sphincter cells. This study was conducted to explore that which of four subtypes of guanine nucleotide-binding protein play an important role in the sling fibers and clasp fibers.
Methods:Thirty-two patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the four subtypes G protein antibodies before agonists were used. The original length and length changes of all kinds of muscle cells to different treatments were recorded using a technique for measuring in vitro cell length of smooth muscle tissue. The maximal dose of acetylcholine, bombesin and substance P were used to induce contraction. All data were presented as x±s and analyzed using SPSS 13.0 statistical program. A P value less than 0.05 was considered statistically significant.
Results: In the cells of the circular layers of the esophagus and the clasp fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gi3 antibodies (P < 0.001 by ANOVA), but not by Go, Gq/11 antibodies, which indicated that, in the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. While in the cells of the circular layers of the gastric fundus and the sling fibers, the contraction induced by acetylcholine, bombesin or substance P was inhibited by Gq/11 antibodies (P < 0.001 by ANOVA) but not by Go, Gi3 antibodies, which indicated that, in the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11.
Conclusion:In the circular layers of the esophagus and the clasp fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gi3. In the circular layers of the gastric fundus and the sling fibers, contraction induced by acetylcholine, bombesin or substance P depends on Gq/11. Guanine nucleotide-binding proteins linked with acetylcholine, bombesin or substance P receptors are unique and muscle-type dependent.
Part 3 The role of phospholipases on the contraction of the human LES’sling fibers and clasp fibers
Objective:Activation of the phospholipases is responsible for hydrolysis of lipid pool and production of second messengers after guanine nucleotide-binding proteins are activated by stimulation with a maximally effective dose of Ach, bombesin or substance P. Which kind of phospholipases is activated in human lower esophageal sphincter’s sling fibers and clasp fibers, and the circular layers of the esophagus and gastric fundus when stimulated by a maximally effective dose of Ach, bombesin or substance P is still not certain. This study was conducted to explore that the role of PC-PLC, PI-PLC, and PC-PLD on the human LES’sling fibers and clasp fibers.
Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and Octomber 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of permeabilized smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. The permeabilized cells were incubated in each of the three phospholipases before agonists were used. When inhibitors (10-6M U-73122, 10-4M propranolol, 10-4M D609 and combination of propranolol and D609) were used, the cells were incubated in the antagonists for 5 min before addition of agonist, either acetylcholine, bombesin or substance P.
Results: (1) PI-PLC antagonist U-73122 (10-6 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the gastric fundus and the sling fibers, but not of the cells of the circular layers of the esophagus and the clasp fibers. The differences among four groups are significant (P<0.05). Which indicated that, PI-PLC played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the gastric fundus and the sling fibers. (2) Conversely, the PC- PLC inhibitor D609 (10-4 M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). Which indicated that, PC-PLC plays an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers. (3) PC-PLD inhibitor propranolol (10-4M) inhibited acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, but not of the cells of the circular layers of the gastric fundus and the sling fibers. The differences among four groups were significant (P<0.05). which indicated that, PC-PLD played an important role in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and the clasp fibers.(4) In acetylcholine, bombesin or substance P -induced contraction of the cells of the circular layers of the esophagus and the clasp fibers, the inhibitory effects of D609 and propranolol were additive. (P=0.014,P=0.027)by paired t test against D609 or propranolol alone.
Conclusion:PC-PLC, PI-PLC, and PC-PLD play different roles in contraction induced by acetylcholine, bombesin or substance P in the circular layers of the esophagus and gastric fundus, and the sling fibers and clasp fibers. The contraction of four muscle type cells in response to acetylcholine, bombesin or substance P is differentially inhibited by different phospholipase specific inhibitors. PI-PLC antagonist inhibits the contraction of the cells of the circular layers of the gastric fundus and the sling fibers; PC-PLC or PLD antagonists inhibits the contraction of the cells of the circular layers of the esophagus and the clasp fibers. And the inhibitory effects result in almost complete abolition of contraction.
PART 4 The role of calmodulin-dependent and PKC-dependent pathways on the contraction of the human LES’sling fibers and clasp fibers
Objective:To investigate the roles of two distinct intracellular signal transduction pathways: Protein Kinase C (PKC)-dependent and calmodulin- dependent, in the contractile mechanism of the clasp and sling fibers, the two parts of human lower esophageal sphincter, in comparison with circular muscle layers of gastric fundus and esophagus. Meanwhile the roles of extracellular and intracellular Ca2+ in this process were studied.
Methods: Five patients undergoing subtotal esophagectomy for middle thoracic esophageal carcinoma in the Fourth Hospital, Hebei Medical University between March 2008 and October 2009 were selected, from which the smooth muscles of the sling and clasp fibers of LES, and the circular layers of the esophagus and gastric fundus were obtained. Suspension of smooth muscle cells from the sling fibers and clasp fibers and the circular layers of the esophagus and gastric fundus were prepared. When Protein Kinase C(PKC)-dependent pathway inhibitors H7, calmodulin-dependent pathway inhibitor CGS9343B, Ca2+-free medium or substitution of 4mmol/L Sr2+ for Ca2+ were used, the cells were incubated in appropriate solution for 5 min before addition of agonists. The length of cells was measured by video-based motion edge-detection system.
Results: Contraction of the cells of the clasp muscles and the esophagus was blocked by H7 and by incubation in Ca2+-free medium, but not by the CGS9343B and 4mmol/L Sr2+. By contrast, contraction of the cells from sling and gastric fundus was blocked only by CGS9343B and 4mmol/L Sr2+.
Conclusion: Contraction of the sling fiber and the circular muscle of the gastric fundus depends on release of intracellular calcium and activation of a calmodulin-dependent pathway. While contraction of the clasp fiber and the circular muscle of the esophagus depends on influx of extracellular calcium and activation of a PKC-dependent signal transduction pathway.
引文
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2 Sigala S, Missale G, Raddino R, et al. Opposing roles for D-1 and D-2 dopamine receptors in the regulation of lower esophageal sphincter motility in the rat. Life Sciences, 1994, 54(15): 1035~1045
3 Preiksaitis HG, Tremblay L, Diamant NE. Cholinergic mechsanism in the cat lower esophageal sphincter show regional variation in vivo and in vitro. Gastroenterology, 1994, 106(2): 381~388
4 Sigala S, Missale G, Missale C, et al. Different neurotransmitter systems are involved in the development of esophageal achalasia. Life Sciences, 1995, 56(16): 1311~1320
5 Preiksaitis HG, Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol, 1997, 272(6 Pt 1): G1321-1327
6 Yuan S, Brookes SJ. Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol, 1999, 412: 669~680
7 Harnett KM, Cao W, Kim N, et al. Signal transduction in esophageal and LES circular muscle contraction. Yale J Biol Med, 1999, 72: 153~168
8 Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physio, 2001, 281(6): G1460~G1467
9 Tian ZQ, Liu JF, Wang GY, et al. Response of human clasp and sling fibers to neuromimetics. J Gastroentrol Hepatol, 2004, 19(4): 440~447
10 Logothetis DE, Kurachi Y, Galper J, et al. Theβγsubunits of GTP-binding proteins activate the muscarinic K channel in heart. Nature 1987, 325:321~326
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2 Sigala S, Missale G, Raddino R, et al. Opposing roles for D-1 and D-2 dopamine receptors in the regulation of lower esophageal sphinctermotility in the rat. Life Sciences, 1994, 54(15): 1035~1045
3 Preiksaitis HG, Tremblay L, Diamant NE. Cholinergic mechsanism in the cat lower esophageal sphincter show regional variation in vivo and in vitro. Gastroenterology, 1994, 106(2): 381~388
4 Sigala S, Missale G, Missale C, et al. Different neurotransmitter systems are involved in the development of esophageal achalasia. Life Sciences, 1995, 56(16): 1311~1320
5 Preiksaitis HG, Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol, 1997, 272(6 Pt 1): G1321-1327
6 Yuan S, Brookes SJ. Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol, 1999, 412: 669~680
7 Harnett KM, Cao W, Kim N, et al. Signal transduction in esophageal and LES circular muscle contraction. Yale J Biol Med, 1999, 72: 153~168
8 Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physio, 2001, 281(6): G1460~G1467
9 Tian ZQ, Liu JF, Wang GY, et al. Response of human clasp and sling fibers to neuromimetics. J Gastroentrol Hepatol, 2004, 19(4): 440~447
10 Sohn UD, Harnett KM, De Petris G, et al. Distinct muscarinic receptors, G proteins, and phospholipases in esophageal and lower esophageal sphincter (LES) circular muscle. J. Pharmacol. Exp. Ther. 267: 1205-1214, 1993
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16 Wess J.G-protein-coupled receptors:molecular mechanisms involved in receptor activation and selectivity of G-protein recognition.FASEB J,1997,11:346~354
17 Conklin BR,Bourne HR.Structural elements of Gαsubunits that interact with Gβγ,receptors,and effectors. Cell,1993,73:631~641
18 Unemori H, Inoue T, Kume S, et al.Activation of G protein Gq/11 through tyrosine phosphorylation of theαsubunit. Science, 1997, 27 6:1878~1881
19 Barr A J,Brass LF,Manning DR.Reconstitution of receptors and GTP-binding regulatory proteins(G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor G protein coupling. J Biol Chem, 1997, 272:2223~2229
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27 Lee CH,Katz A,Simon MI.Multiple regions of Gα1contributes to the specificity of activation by the C5 receptor.Mol Pharmacol,1995, 47:218~223
28 Mazzoni MR,Hamm HE. Interaction of transduction witlight activated rhodopsin protects it from proteolytdigestion by trypsin. J Biol Chem, 1996,271:30034~30040
29 Logothetis DE, Kurachi Y, Galper J, et al. Theβγsubunits of GTP-binding proteins activate the muscarinic K channel in heart. Nature 1987, 325:321~326
30 Liu JF, Gao LP, Wen SW, et al. Responses of human sling and clasp fibers to cholecystokinin and gastrin through CCK receptors. J Gastroenterol Hepatol 2008;23:1608-1612
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1 Richardson BJ, and Welch RW. Differential effect of atropine on rightward and leftward lower esophageal sphincter pressure. Gastroenterology, 1981, 81: 85~89
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4 Sigala S, Missale G, Missale C, et al. Different neurotransmitter systems are involved in the development of esophageal achalasia. Life Sciences, 1995, 56(16): 1311~1320
5 Preiksaitis HG, Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol, 1997, 272(6 Pt 1): G1321-1327
6 Yuan S, Brookes SJ. Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol, 1999, 412: 669~680
7 Harnett KM, Cao W, Kim N, et al. Signal transduction in esophageal and LES circular muscle contraction. Yale J Biol Med, 1999, 72: 153~168
8 Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physio, 2001, 281(6): G1460~G1467
9 Tian ZQ, Liu JF, Wang GY, et al. Response of human clasp and sling fibers to neuromimetics. J Gastroentrol Hepatol, 2004, 19(4): 440~447
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12 Biancani P, Harnett KM, Sohn UD, et al. Differential signal transduction pathways in cat LES tone and response to ACh. Am J Physiol, 1994, 266: 767-774
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3 Preiksaitis HG, Tremblay L, Diamant NE. Cholinergic mechsanism in the cat lower esophageal sphincter show regional variation in vivo and in vitro. Gastroenterology, 1994, 106(2): 381~388
4 Sigala S, Missale G, Missale C, et al. Different neurotransmitter systems are involved in the development of esophageal achalasia. Life Sciences, 1995, 56(16): 1311~1320
5 Preiksaitis HG, Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol, 1997, 272(6 Pt 1): G1321-1327
6 Yuan S, Brookes SJ. Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol, 1999, 412: 669~680
7 Harnett KM, Cao W, Kim N, et al. Signal transduction in esophageal and LES circular muscle contraction. Yale J Biol Med, 1999, 72: 153~168
8 Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physio, 2001, 281(6): G1460~G1467
9 Tian ZQ, Liu JF, Wang GY, et al. Response of human clasp and sling fibers to neuromimetics. J Gastroentrol Hepatol, 2004, 19(4): 440~447
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17 Conklin BR,Bourne HR.Structural elements of Gαsubunits that interact with Gβγ,receptors,and effectors. Cell,1993,73:631~641
18 Unemori H, Inoue T, Kume S, et al.Activation of G protein Gq/11 through tyrosine phosphorylation of theαsubunit. Science, 1997, 27 6:1878~1881
19 Barr A J,Brass LF,Manning DR.Reconstitution of receptors and GTP-binding regulatory proteins(G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor G protein coupling. J Biol Chem, 1997, 272:2223~2229
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29 Logothetis DE, Kurachi Y, Galper J, et al. Theβγsubunits of GTP-binding proteins activate the muscarinic K channel in heart. Nature 1987, 325:321~326
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1 Richardson BJ, and Welch RW. Differential effect of atropine on rightward and leftward lower esophageal sphincter pressure. Gastroenterology, 1981, 81: 85~89
2 Sigala S, Missale G, Raddino R, et al. Opposing roles for D-1 and D-2 dopamine receptors in the regulation of lower esophageal sphincter motility in the rat. Life Sciences, 1994, 54(15): 1035~1045
3 Preiksaitis HG, Tremblay L, Diamant NE. Cholinergic mechsanism in the cat lower esophageal sphincter show regional variation in vivo and in vitro. Gastroenterology, 1994, 106(2): 381~388
4 Sigala S, Missale G, Missale C, et al. Different neurotransmitter systems are involved in the development of esophageal achalasia. Life Sciences, 1995, 56(16): 1311~1320
5 Preiksaitis HG, Diamant NE. Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction. Am J Physiol, 1997, 272(6 Pt 1): G1321-1327
6 Yuan S, Brookes SJ. Neuronal control of the gastric sling muscle of the guinea pig. J Comp Neurol, 1999, 412: 669~680
7 Harnett KM, Cao W, Kim N, et al. Signal transduction in esophageal and LES circular muscle contraction. Yale J Biol Med, 1999, 72: 153~168
8 Muinuddin A, Xue S, Diamant NE. Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation. Am J Physiol Gastrointest Liver Physio, 2001, 281(6): G1460~G1467
9 Tian ZQ, Liu JF, Wang GY, et al. Response of human clasp and sling fibers to neuromimetics. J Gastroentrol Hepatol, 2004, 19(4): 440~447
10 Sohn UD, Chiu TT, Bitar KN, et al. Calcium requirements for acetylcholine-induced contraction of cat esophageal circular muscle cells. Am J Physiol Gastrointest Liver Physiol, 1994, 266: 330–338
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