| The lower esophageal sphincter (LES), which is located at thegastroesophageal junction in human, is a specially differentiated thickenedregion of the circular muscle layer, extending over an axial distance about2–3cm. In1979, Liebermann-Meffer first proposed that the human LES consistsof clasp fibers and sling fibers at the lesser curvature and the greater curvaturerespectively. The crural diaphragm, the sling fibers and the clasp fibers are ina state of constant contraction, which is due to both neurogenic and myogenicregulation of the human lower esophageal sphincter and forms a high pressurezone at the esophageogastric junction (EGJ) to prevent the gastric contentsreflux into the esophagus.In the human lower esophageal sphincter, the functional regulation ofcontraction and relaxation is completed by several neurotransmitters,hormones and spontaneous myogenic factors, which are under the control ofthe the central nervous system. Previous studies revealed that the vagalefferent fibers synapse and the enteric motor neurons (EMN), which arelocated in the myenteric plexus (MP), form the inhibitory and excitatorymovement pathway. The preganglionic neurons that form the inhibitory andthe excitatory pathway are cholinergic. Moreover, the inhibitorypostganglionic neurons are nonadrenergic noncholinergic (NANC), whereasthe excitatory postganglionic neurons are cholinergic. Furthermore, both theneurotransmission of the excitatory and inhibitory vagal pathways couldrelease a variety of neurotransmitters at and regulate the tone of the humanLES. The activation of the vagal inhibitory pathway can induce relaxation ofthe LES, whereas the activation of the vagal excitatory pathway can induce thecontraction of the LES.Previous researches show that esophageal motor disorders such as achalasia, nutcracker esophagus and diffuse esophageal spasm demonstratedabnormalities of the human LES. Therefore, it is important for the treatmentand diagnosis of esophageal motility disorders to study the regulationmechanism of the human lower esophageal sphincter. The studies showed thatthe regulatory mechanism of the human lower esophageal sphincter involvesof vasoactive intestinal peptide, nitric oxide, acetylcholine, their signaltransduction pathways and receptors.ETs are composed of21-aminoacids produced by endothelial cells andconsidered to be potent vasoconstrictor. Recent studies found that they can notonly induce the angiogenesis, mitosis and differentiation, but also act the roleof cytokines. Previous studies have identified three isoforms of ET (ET-1,ET-2and ET-3), which are involved in a variety of physiological andpathological processes. ET-1is most potent one among ETs, whereas ET-3isthe weakest once. ET peptides exhibit their functions in a number ofmammalian physiological systems, including gastrointestinal. ETs act via twodistinct isoforms of receptors in human body, i.e. ETA and ETB, which can befound expressed in many mammalian gastrointestinal tract.Firstly, this study used the quantitative polymerase (quantitative PCR) toidentify the relative quantity of each ETR expression. Secondly, reversetranscription-polymerase chain reaction (RT-PCR) and western blotting wereused to verify the conclusions. Finally, measurement of muscle tension in vitro,and electrical field stimulation (EFS) were used to identify the function of theETRs in the human LES. The purpose of present study is to investigate theexpression of endothelin receptors and the role that the endothelin receptorsplay in modulating human lower esophageal sphincter function. In this way,we could further demonstrate the regulatory mechanism of the human loweresophageal sphincter in detail, and propose the theoretical bases for themedication of esophageal motility disorders much more properly.PartⅠ Expression of endothelin receptors in human lower esophagealsphincterObjective: Endothelin receptors (ETRs) belong to the family of the G protein-coupled receptors, mediate the physiological functions of thenonadrenergic noncholinergic (NANC) neurotransmitter endothlin. We usequantitative polymerase chain reaction (quantitative PCR) to identify therelative quantity of each ETR expression in four muscle strips including slingfibers, clasp fibers, circular muscle strips of esophagus and gastric foundus.Then, reverse transcription-polymerase chain reaction (RT-PCR) and westernblotting were used to verify the conclusions, in order to test whether theexpressions of ETRs mRNAs are consistent with the distribution of the ETRsproteins and to investigate the role of ETRs in modulating human LESfunction.Methods: The specimens are obtained from32patients, who underwentesophagectomy for mid-third esophageal carcinoma in the Department ofThoracic Surgery, the Fourth Hospital, Hebei Medical University, betweenDecember2011and November2012. There were7females and23males,with a mean age of61years. Each specimen including part of thegastroesophageal junction, the esophageal body, and the gastric fundus, wasresected en bloc in the operating room. The submucosa and mucosa wereremoved from the specimens, by sharp dissection. The muscle strips of claspand sling fibers of LES, and circular muscle strips of gastric foundus andesophagus were obtained from different positions of the gastroesophagealjunction (EGJ) and adjacent tissues. In laboratory, total RNA was extracted byacid guanidinium thiocyanate-phenolchloroform extraction. Before theexperiment, identify its integrity and purity with the ultravioletspectrophotometer and the electrophoresis in1%agarose gels successively.Then quantitative polymerase chain reaction (quantitative PCR) wasperformed to determine the relative quantities of each ETR expression, at first.Secondly, reverse transcription-polymerase chain reaction (RT-PCR) wasperformed to determine the mRNA expressions of ETRs. Each integratedoptical density (IOD) of the amplified products electrophoresis bands wascalculated with Gel-Pro software. The expressions of ETRs mRNA weredemonstrated by the ratio of IOD value of ETRs bands to β-actin band. The integral membrane protein receptors, which were extracted from muscle strips,were quantitated and adjusted to the identical concentration. Then, differentETRs proteins were separated by electrophoresis. Using different polyclonalantibody of each ETR after the trarsmembrane, each protein expression wasdetected by ultraviolet detector. The IOD values were calculated with theOddessy software.Results: The total RNA value of A260/280, which was determined bythe ultraviolet spectrophotometry, was between1.8and2.0. The brightnessand width of18S band was one half than28S band in1%agarose gels.Quantitative PCR identified that the band of β-actin mRNA was uniformly130bp in each strip amplified product of the specimen. After calculating thedata, we found that each ETR mRNA expression was identified in eachmuscle strip of the specimen. Next, dyed with5Xbuffer, the samples wereelectrophoresed in agarose gel, in order to verify whether the product wascorrect. After the ultraviolet detection, the band of ETA and ETB was166bpsand128bps respectively, consistent with the expected size. It was found thatthe ETA expression is more than the ETB by calculation. The ETA expressionrelative quantities of the four muscle strips including clasp fibers, sling fibers,circular muscle strips of esophagus and gastric foundus were0.01151±0.00055,0.01207±0.00086,0.01185±0.00031and0.01161±0.00055respectively. The ETB expression relative quantities of the four muscle stripsincluding clasp fibers, sling fibers, circular muscle strips of esophagus andgastric foundus were0.001691±0.00020,0.001793±0.00030,0.001616±0.00028and0.001571±0.00013respectively. Each amplifiedproduct of the quantitative PCR was consistent with the expected size, whichwas designed before the experiment. Comparing the expression of differentETR mRNA in the same muscle strips, significant differences were found(F=72.291; P=0.000). However, there was no significant difference in ETRsmRNA expression among the four muscle strips (F=0.398; P=0.5329). Then,the RT-PCR was performed to verify the conclusion mentioned above. Wefound that the band of β-actin mRNA was uniformly362bp in each strip of the specimens, after electrophoresis. The band of each ETA and ETB wasconsistent with the expected size including263bps and200bps respectively.The ETA expression relative quantities of the four muscle strips includingclasp fibers, sling fibers, circular muscle strips of esophagus and gastricfoundus were0.01163±0.00062,0.01219±0.00049,0.01147±0.00049and0.01148±0.00054respectively. The ETB expression relative quantity is lessthan the ETA. The ETB expression relative quantities of the four muscle stripsincluding clasp fibers, sling fibers, circular muscle strips of esophagus andgastric foundus were0.001725±0.00019,0.001700±0.00029,0.001632±0.00027and0.001625±0.00020respectively. Comparing the expression ofdifferent ETR mRNA in the same muscle strips, significant differences werefound (F=102.827; P=0.000). However, there was no significant difference inETRs mRNA expression among the four muscle strips (F=0.521; P=0.476).Protein expression of each ETR subtype was determined, they were ETA andETB, with respective molecular,48.73kDa and50kDa. In the same musclestrip, there was a significant difference in IOD values for different ETRS(F=533.841; P=0.000). There was no significant difference in IOD valuesamong the four muscle strips.(F=0.398; P=0.755).Conclusion: ETA and ETB can be identified in the human loweresophageal sphincter, the rank order of ETR expression is ETA>ETB.Probably these two ETRs contribute to the regulation of human LES function.PartⅡ The role of endothelin receptors in modulating human loweresophageal sphincterObjective: To determin the effects of non-selectinve endothelin receptoragonist and selective endothelin receptor antagonists on the LES complexmuscle strips, and investigate the role of endothelin receptor subtypes inregulating relaxation and contraction of the human LES.Methods: The specimens were abtained from30patients who underwentesophagectomy for mid-third esophageal carcinoma in the Department ofThoracic Surgery, the Affiliated Hospital, Hebei University, from October2012to December2013in this study. There were14females and16males, with a mean age of65years. After surgical excision in the operating room,each specimen was resected en bloc and was placed immediately in4℃Krebs solution in the laboratory successively. If there were any part of thetissue prepared for experiment contained macroscopically visible tumour,specimen was not included in this study. In the laboratory, specimens werefixed on a wax plate, which contained TBS, bubbled with continuous mixedgas of95%O2and5%CO2, washed with37℃Krebs solution, with themucosal surface facing upside to sustain its approximate in situ dimensions.Then the submucosa and mucosa were removed by sharp dissection gently.There was a thickened band of cirlular smooth muscle at the esophageogastricjunction (EGJ), namely the clasp and sling fibers of LES, adjacent to thelesser and greater curvature of the stomach, respectively. The clasp and slingmuscle strips of LES were prepared by the method described previously. Theclasp fibers, sling fibers were separated and prepared into (8~12) mm×(2~4)mm muscle strips.With each end tied with silk, the muscle strips were placed in bath, whichwas bubbled with continuous mixed gas (5%CO2and95%O2) and containinga10ml Krebs solution maintained37℃. The upper end of the muscle stripwas fixed to JZ101(an isometric force transducer) and the tension changes ofthe muscle strips was recorded by the software of MedLab6.0. The musclestrips were stretched gently and slightly to make the tension of the musclestrips maintaining at400mg, then the length of the muscle strips at thattension is the initial length L0. Then sequentially, at the increments of25%L0each time, the muscle strips were stretched to200%L0, which was recordedas most suitable initial length.The muscle strips with the most suitable initial length were equilibratedfor40minutes, then add10-6mol/L Carbachol into the bath, and record thetesion data, wash. When the strip was recoverd，then the non-selective agonistof the ETRs, namely ET-1, was added into the bath from10-10to10-6mol/L, with a cumulative manner. Not until the response of the previousconcentration stabilized, successive concentrations of the agonists were added in the bath. The time interval is5minutes. Based on the determined tensiondata above, the cumulative administration concentration-responsedose-response curves were constructed. Next, the antagonist was added30min before adding the agonist to observe the effect. And the concentration ofthe antagonist is same to the agonist, which induced maximum effectproduced by the muscle strips. In this study, we use carbachol(1×10-9mol/L)-induced contraction as a reference to express the contractileresponse to the non-selective agonist.Results:1The non-selective endothelin receptor agonist ET-1could induce thedetectable contraction of the clasp and sling fibers of the human LES at theconcentration of10-9mol/L. The maximal tension of contraction (about58%of the tension induced by1×10-9mol/L carbachol) was produced at theconcentration of10-7mol/L. There was no significant difference between thetension of clasp fibers and sling fibers, caused by ET-1.(F=0.25P=0.62)2The selective antagonist could block the contractile effect produced bythe non-selective endothelin receptor agonist ET-1.(F=67.9P=0.000) Theselective ETA antagonist BQ-123could partly block the contractile effectproduced by the non-selective endothelin receptor agonist ET-1, at theconcentration of (10-9、10-8、10-7、10-6mol/L).(F=31.3P=0.0000) Though theselective ETB antagonist BQ-788could also partly block the contractile effectproduced by the non-selective endothelin receptor agonist ET-1at theconcentration mentioned above (F=5.6P=0.02511), the antagonistic effect ofBQ-788is significantly less than BQ-123.(F=11.5P=0.0021)Conclusion:1The non-selective dopamine receptor agonist ET-1could induce thedetectable contraction of human LES at minimal concentration of10-9mol/L.With the sequential increase of ET-1concentration, the muscle strips graduallyreached the maximum contractile effect. It suggests that endothelin receptor A,and endothelin receptor B may be involved in the contractile regulation of thehuman lower esophageal sphincter. 2The selective ETA antagonist BQ-123and the selective ETB antagonistBQ-788could both partly inhibit the contractile effect produced by thenon-selective endothelin receptor agonist ET-1in human lower esophagealsphincter. The conclusion mentioned above further demonstrate that theendothelin receptor A (ETA) and endothelin receptor B (ETB) are involved inthe regulation of the human lower esophageal sphincter (contractile function)and the contribution of ETA is larger than ETB.PartⅢ The contribution of endothelin receptors in the response ofhuman lower esophageal sphincter under the electical fieldstimulationObjective: To identify the role, which the endothelin receptor antagnostsplay, in regulating the clasp fibers and sling fibers of the human loweresophageal sphincter (LES) under the electical field stimulation (EFS), andinvestigate the effect of endothelin receptors in modulating human LESfunction.Methods: The specimens were abtained from thirty patients whounderwent esophagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Affiliated Hospital, Hebei University,from October2012to December2013in this study. There were9females and21males, with a mean age of61years. Each muscle strip of the LES wasprepared by using same method described previously. With each end tied withsilk, the muscle strips were placed in bath, which was bubbled withcontinuous mixed gas (5%CO2and95%O2) and containing a10ml Krebssolution maintained37℃. The upper end of the muscle strip was fixed toJZ101(an isometric force transducer) and the tension changes of the musclestrips was recorded by the software of MedLab6.0. The muscle strips of LESwere sequentially stretched to most suitable initial length using similarmethods mentioned previously (The difference was that muscle stripsmaintaining at200mg, record the length as the initial length L0) and had a37℃bath for60min in Krebs solution, which bubbled with continuous mixedgas (95%O2and5%CO2). EFS stimulation parameters: single-pulse square wave, pulse frequency1-512Hz, voltage50V, width5ms. After added L-NNA, the muscle strips issubjected to electrical stimulation according to the frequency from small tobig and the maximum effect to EFS was assessed. The muscle strip wasstimulated again after20min of administration of Tetrodotoxin, BQ-123(aselective ETA antagonist). Then, we repeated the procedure and replacedBQ-123with BQ-788(a selective ETB antagonist). The responses in all of theexperiments were quantified based upon a percentage of the baseline value ofmuscle strip tone relative to the nadir of the response. The data wereexpressed as means±standard error.Results:1The EFS could induce a frequency-dependent contraction in the slingfibers and the clasp fibers of human LES, and the maximum contractileelectrical stimulation frequency was128Hz. The maximum contractionpercentage of the lower esophageal sphincter by EFS-induced was about19.3±0.8%.2After the administration of tetrodotoxin, the frequency-dependentcontraction in the sling and clasp fibers of human LES, which was induced byEFS, significantly decreased.(F=23.3P=0.000)3The selective antagonist BQ-123(F=0.78P=0.384661) and BQ-788(F=0.38P=0.542583) could not inhibit the contraction of human LES musclestrips, which was induced by EFS. There was no significant differencebetween before and after administration.Conclusion:1After the administration of tetrodotoxin, the frequency-dependentcontraction in the sling and clasp fibers of human LES, which was induced byEFS, was inhibited. There was significant difference between the contractionof the muscle strips before and after the administration of tetrodotoxin. Theoptimum frequency leading to maximum contraction was128Hz. It suggeststhat the frequency-dependent contraction of LES induced by EFS is nerve origin.2The selective antagonist BQ-123and BQ-788could not inhibit thefrequency-dependent contraction induced by the EFS in the sling fibers andthe clasp fibers of human LES.(BQ-123F=0.78P=0.384661, BQ-788F=0.38P=0.542583) It suggests that the ETA and ETB was note involed inthe frequency-dependent contraction induced by EFS, and ETA, ETB werenot involved in the nerve regulation. |
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