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Serotonin Receptors Expression And Function In Human Lower Esophageal Sphincter

Posted on:2016-09-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:H F LiFull Text:PDF
GTID:1224330461962847Subject:Clinical Medicine
Abstract/Summary:PDF Full Text Request
The human lower esophageal sphincter(LES) is located at the esophagogastric junction(EGJ) circular muscle bundle of partial thickening. Liebermann-Meffert proposed this concept, and points out that the human LES consists of clasp fibers at the lesser curvature and sling fibers at the greater curvature. The clasp fibers, sling fibers and the diaphragm build up the HPZ(high pressure zone). In swallowing the LES prevent reflux of gastric contents, when eating the relaxation of the LES allow food enters the stomach cavity.The mechanism of relaxation and contraction of the LES is regulated by neurotransmitters, spontaneous myogenic factors and several hormones in the control of the central nervous system(CNS). The output nerve ending of the vagus nerves and the intestinal motor neurons of the LES constitute the excitatory and inhibitory movement pathway. The excitatory and inhibitory pathway could adjust the relaxation and contraction of the LES. The activation of the excitatory neurotransmitter release Ach and substance P could increase the lower esophageal sphincter pressure. The activation of the inhibitory neurotransmitter release nitric oxide and vasoactive intestinal peptide could decrease the lower esophageal sphincter pressure. The sympathetic nerve involved in the regulation of lower esophageal sphincter located in the T6-T10 parts of spinal cord. The preganglionic nerves are cholinergic and target the celiac ganglia to sensitive noradrenergic postganglionic fibers that directly innervate the lower sphincter smooth muscles or synapse onto the enteric motorius.Research shows that esophageal motor disorders such as diffuse esophageal spasm, gastroesophageal reflux disease, achalasia and the nutcracker esophagus and other diseases pathogenesis were all associated with dysfunction of lower esophageal sphincter. Therefore, the research on functions of lower esophageal sphincter has significance for the diagnosis and treatment of esophageal motility disorders. The domestic and abroad researches demonstrated that the regulatory mechanism of LES involves receptors, signal transduction pathways and neurotransmitters.5-hydroxytryptamine(5-HT) also known as serotonin, as a neurotransmitter and vasoactive substances are widely distributed in the central nervous system and peripheral tissues. 5-HT played an important role in feeling, emotion, cardiovascular function and breathing, appetite. 5-HT as a neurotransmitter in central nervous system is mainly exist in the pineal gland and hypothalamus, involved in pain, sleep, temperature and other physiological regulation. The content and function abnormal of 5-HT are associated with mental illness and other diseases. On the other hand, 5-HT also plays an important role in the gastrointestinal movement, sensory and secretory by acting on different receptors. 5-HT receptor has identified 7 types and further subdivided into no less than 16 subtypes. 5-HT3 receptor is voltage gated ion channel receptor, and the rest are G protein coupled receptors(G protein coupled receptors, GPCRs).The expression of 5-HT receptors were detected by means of the technology of RT-PCR, real-time quantitative polymerase chain reaction(q PCR) and western blotting. In the further experiment, measurement of fresh isolated muscle tension and electrical field stimulation(EFS) were used to detect function of 5-HT receptors in human LES. The study has laid a foundation for further study of 5-HT receptor in the human LES functional regulation, so as to provide an important basis for the explanation of human lower esophageal sphincter mechanism regulation and treatment of esophageal motor dysfunction diseases. Part Ⅰ Expression of 5-HT Receptors in the Human Lower Esophageal SphincterObjective: In this part of experiment, we first identified the expression of 5-HT receptors m RNA in four muscle strips including clasp fibers, sling fibers, circular muscle strips of the esophagus and stomach by reverse transcription-polymerase chain reaction(RT-PCR) and real-time quantitative polymerase chain reaction(q PCR). Then the western-blot analysis was used to study the expression of 7 receptor protein in the four muscle strips.Methods: The muscle strip specimens were collected from 27 patients who underwent esphagectomy at the Fourth Hospital of Hebei Medical University from January 2012 to May 2012. There were 16 male and 11 female, with an average age of about 57 years. Fresh esophageal and gastric specimens were removed from patients in the operation room. The mucosa and submucosa tissues were completely removed and pay attention to the protection of intact muscular layer with a scalpel. The lower esophageal sphincter is an thickened circular muscle layer near the connecting part of the remote end of esophagus and near the end of stomach. We define the clasp fibers at the lesser curvature and sling fibers at the greater curvature. We define the incrassated circular muscle bundles as clasp and sling fibers in the positin above. Then the muscle strips for the circular muscles of esophageal body and gastric fundus, clasp fibers, sling fibers were prepared for the next step. The tissues should be abandoned if it were invaded by tumor. Reverse transcription-polymerase chain reaction(RT-PCR) was performed using primers designed to accord with the respectively m RNA of seven 5-HT receptors. After determining the m RNA were expressed in four muscle strips, then apply the real-time quantitative PCR(q PCR) method to determine the relative content. Total proteins were extracted and then adjusted to the same concentration. First electrophoretic separation of the seven 5-HT receptors, then they were transferred onto a polyvinylidene difluoride. After that, using specific 5-HT receptors’ polyclonal antibody to detect the protein expression. The result was demonstrated using the infrared imaging system. Gel Pro software was used to calculate the integrated optical density(IOD) of every receptor.Results: The purity of RNA abstracted from muscle strip meet the requirement of test. The band of β-actin m RNA has uniform brightness at 540 bp and the PCR product was in agreement with the expected length. Transcripts for seven 5-HT receptors were identified in the various muscle strips and the length of the amplification product was consistent with the designed length. There was significant differences between the expression of different 5-HT receptors’ m RNA in the same muscle strips(F=81.281; P=0.000). The expression level of different 5-HT receptors was: 5-HT3AR=5-HT4R>5-HT1AR=5-HT2AR>5-HT5AR=5-HT6R=5-HT7 R. There was no significant difference in m RNA expression of the same 5-HT receptor between four muscle strips(F=0.218; P=0.695). Protein expression of some 5-HT receptor subtypes were detected: 5-HT1 AR, 5-HT2 AR, 5-HT3 AR, 5-HT4 R and 5-HT7 R. The five receptor subtypes molecular was 48 KD, 46 KD, 53 KD, 55 KD, 44 KD and 54 KD. The protein of 5-HT5 and 5-HT6 receptor was not detected. There was a significant difference in relative expression level for five 5-HT receptors in the same muscle strip(F=648.269; P=0.000). The rank order of the five receptor protein relative expression level was 5-HT3AR= 5-HT4R> 5-HT1AR=5-HT2AR>5-HT7 R. There was no significant difference in relative expression level between the four muscle strips about the same receptor(F=0.194; P=0.801).Conclusion: There are seven kinds of 5-HT receptor subtypes in the human LES. Their relative expression levels of m NRA is 5-HT3AR= 5-HT4R>5-HT1AR=5-HT2AR>5-HT5AR=5-HT6R=5-HT7 R. The protein of 5-HT5 and 5-HT6 receptor was not detected in the human LES, the rest of the receptor protein expression levels is 5-HT3AR=5-HT4R> 5-HT1 AR = 5-HT2AR>5-HT7 R. PartⅡ The Role of 5-HT Receptors in Modulating Human Lower Esophageal SphincterObjective:To investigate the effects of selective and non-selective 5-HT receptors agonists and antagonists on the clasp and sling fibers, and explore the role of 5-HT receptors in adjusting the function of relaxation and contraction in LES.Methods: The experiment muscle strips were collected from 29 patients who underwent esphagectomy in the Department of Thoracic Surgery at the Fourth Hospital, Hebei Medical University from April 2012 to October 2012. There were 15 males and 14 females, with the average age of 59 years. Esophagogastric connection specimens were chipped and preserved in the 4 ℃ Krebs. First the specimens were washed with 37℃ Krebs solution, then pined on a wax plate maintain Krebs, with gas of 95% O2 and 5% CO2 was ventilated. The mucosa and submucosa were eliminated with a scalpel. The clasp and sling fibers should be confirmed as thickened circular oriented muscle in the gastric cardia. The clasp and sling fibers were prepared with 2mm×10mm muscle strips. Each end of the muscle strip was fastened and placed in a 10 ml bath filled with Krebs liquid with continuous mixed gas of 95% O2 and 5% CO2. The tenesion changes of the muscle strips was recorded by Medlab signal acquisition application. The muscle strips were drew softly until the tension reach 200 mg. The length of the muscle strips at the tension was named as the initial length( L0). The muscle strips were repeat drew and once stretch about 25% of the L0, until the strips run up to 200% of the initial length as the optimum initial length. The optimum initial muscle strips were stabilized 30 minutes, then the non-selective 5-HT agonists were added into the thermostatic groove with the manner from 10-9 to 10-3 mol/L. Each concentration of the agonist should be added until the reaction of the previous agonist concentration achieved the maximum reaction. The concentration-response dose-response curves were built based on the outcomes. The antagonist was added before adding the agonist to observe the effect of the antagonist effect. The concentration of the antagonist is same to the the agonist. All of the responses with the experiments were quantified based on a percentage of the basal value of muscle strip tone relative to the nadir of the response.Results:1 Effect of non-selective 5-HT receptor agonist 5-HT and antagonist methysergide maleate on the human LESThe non-selective 5-HT receptor agonist 5-HT could induce contraction of the clasp and sling fibers at the concentration from 10-9 to 10-3mol/L. There was no significant difference in the response to 5-HT between clasp and sling fibers(P=0.67). The response induced by 5-HT could be inhibited by non-selective 5-HT receptor antagonist methysergide maleate.2 Effect of selective 5-HT3 receptor agonist 2-Methyl-5-HT and antagonist granisetron on the human LESThe selective 5-HT3 receptor agonist 2-Methyl-5-HT induced a concentration-dependent contraction of the clasp and sling fibers at the concentration from 10-9 to 10-3mol/L. The optimal concentration was 10-4mol/L and could cause maximum contraction percentage. The maximum contraction percentage of the clasp was(35±5.8)%. The maximum contraction percentage of the sling was(37±6.7)%. There was no significant difference in contraction response between them(P=0.63). Granisetron could inhibit the contraction of clasp and sling induced by 2-Methyl-5-HT and there was significant difference in contraction response before and after administration( P<0.01). The maximum contraction of clasp was(8.4±1.5)% after joined granisetron, and the sling was(9.1±1.2)% after joined granisetron.3 Effect of selective 5-HT4 receptor agonist tegaserod and antagonist GR113808 on the human LESThe selective 5-HT4 receptor agonist tegaserod induced a concentration-dependent contraction of the clasp and sling fibers at the concentration from 10-9 to 10-3mol/L. The optimal concentration was 10-4mol/L and could cause maximum contraction percentage. The maximum contraction percentage of the clasp was(44±7.7)%. The maximum contraction percentage of the sling was(46±8.3)%. There was no significant difference in contraction response between them(P=0.52). GR113808 could inhibit the contraction of clasp and sling induced by tegaserod and there was significant difference in contraction response before and after administration(P<0.01). The maximum contraction of clasp was(9.2±1.8) % after joined GR113808, and the sling was(10.1±1.5)% after joined GR113808.4 Effect of selective 5-HT7 receptor agonist LP-44 and antagonist SB26997 on the human LESThe selective 5-HT7 receptor agonist LP-44 and antagonist SB26997 had no effect on the clasp and sling fibers. There was no response of contraction or relaxation after added with LP-44 or SB269970 in the LES fibers.Conclusions:1 The non-selective 5-HT receptor agonist 5-HT could induce contraction of clasp and sling fibers. The non-selective 5-HT receptor antagonist methysergide maleate could inhibit the contraction response induced by 5-HT. This part of study research indicated that the effect of 5-HT on the reaction of LES is achieved through 5-HT receptor.2 The selective 5-HT3 receptor agonist 2-Methyl-5-HT and the selective 5-HT4 receptor agonist tegaserod could induce concentrationdependent contraction of the clasp and sling fibers. The selective 5-HT3 receptor antagonist granisetron and the selective 5-HT4 receptor antagonist GR113808 could inhibit the contraction induced by 2-Methyl-5-HT and tegaserod. The fact indicated that the 5-HT3 receptor and 5-HT4 receptor involved in the contraction response of the human LES.3 The selective 5-HT7 receptor agonist LP-44 and antagonist SB26997 had no effect on the human LES. This indicated 5-HT7 receptor may be did not involved in the function regulation of human LES. Part Ⅲ The Contribution of 5-HT Receptors in the Response ofHuman Lower Esophageal Sphincter Under the Electical Field StimulationObjective: To identify the the role of 5-HT receptors in adjusting human lower esophageal sphincter(LES) under the electical field stimulation(EFS), and investigate the role of 5-HT receptors in the function of human LES.Methods: The experiment muscle strips were collected from 25 patients who underwent esphagectomy for mid-third esophageal cancer in the Department of Thoracic Surgery at the Fourth Hospital, Hebei Medical University from August 2012 to November 2012. There were 13 males and 12 females, with the average age of 62 years. The clasp and sling fibers were get ready using the methods that were described in part II. The fresh clasp and sling fibers were collected in the operating room and were placed in a 10 ml thermostatic bath(37°C) containing Krebs solution. The Krebs liquid ventilated with 5% CO2 and 95% O2 mixed gas. The upper of the strip was tied with tension transducer and the lower was fastened to the L-shaped bracket with platinum electrode, and the Medlab signal acquisition was used to record muscle strip tension changes of every muscle strip. The muscle strips were in the situation of optimum initial length. All the muscle strips were make sured in the middle of the platinum electrode and the range between the muscle strips and the electrode was more than 3 mm. The EFS stimulation parameters: single-pulse square wave, pulse width 5ms, voltage 50 V, frequency 1-512 Hz. Electrical stimulation was conducted on the basis of the frequency from small to large and the maximum reaction after stimulation was recorded. EFS stimulation parameters: single-pulse square wave, pulse width 5ms, voltage 50 V, frequency 1-512 Hz. After EFS stimulation and the muscle strip was in balance, then added Tetrodotoxin(TTX). EFS was conducted again after 20 min administered with TTX, and the reaction of muasle strip was compared before and after using TTX. The muscle strip was stimulated after 20 min of administration of selective 5-HT3, 5-HT4 and 5-HT7 receptors antagonists granisetron, GR113808, SB269970 with the concentration of 10-4 mol/L. The responses of all the tests were quantified based on a percentage of the base line value of muscle strip tone relative to the nadir of response. The data were expressed as means±standard error.Results:1 The EFS could induce the clasp and sling fibers to generate a frequency-dependent relaxation response. The optimal frequency initiating the maximum relaxation was 64 Hz. The maximum relaxation percentage of the clasp was(19.1±2.3)% and the sling was(21.3±2.7)%. There was no significant difference in the relaxation responses between the clasp and sling fibers(F=0.21, P=0.66).2 Tetrodotoxin could significantly abated the frequency-dependent relaxation in the LES by EFS-induced. There was significant difference in relaxation of the human LES muscle strips before and after administration.3 The selective 5-HT4 receptor antagonist GR113808 produced significant change in the frequency-dependent relaxation in LES induced by EFS.4 The selective 5-HT3 and 5-HT7 receptor antagonists granisetron and SB269970 did not produce significant change in the frequency-dependent relaxation in LES induced by EFS.Conclusion:1 The EFS could induce frequency-dependent relaxation in the clasp and sling fibers of human LES and the optimal frequency resulting in maximum relaxation is 64 Hz.2 Tetrodotoxin could significantly reduce the frequent-dependent relaxation in the LES by EFS. It is indicate that the frequency-dependent relaxation of LES by EFS is source of neurogenic.3 The 5-HT4 receptor other than 5-HT3 receptor and 5-HT7 receptor may be involved in the the frequency-dependent relaxation of the human LES induced by the EFS.
Keywords/Search Tags:Lower esophageal sphincter, clasp fibers, sling fibers, 5-HT receptor, electrical field stimulation
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