| The esophagogastric junction (EGJ) is a complex valvular structure. The lower esophageal sphincter (LES) is one component of the EGJ that is conceptually a complex unit integrating both LES and diaphragmatic elements. The LES is a special thickened circular muscle layer about2-3cm in human and Liebermann-Meffer proposed firstly that the huanm lower esophageal sphincter consists of sling fibers at the greater curvature and clasp fibers at the lesser curvature in1979. It prevents reflux during nondeglutitive periods and facilitates flow during periods of opening.The mechanism of contraction and relaxation of the LES is regulated by spontaneous myogenic factors, several hormones and neurotransmitters in the control of the central nervous system. The LES is innervated by vagal preganglionic and sympathetic postganglionic efferents. The vagal preganglionic fibers innervate the LES smooth muscle via ganglionic myenteric plexus neurons and provide both inhibitory and excitatory innervation to the LES. The inhibitory and excitatory preganglionic cell bodies are located within the caudal and rostal parts of the dorsal motor nucleus of the vagus, respectively. Typically cholinergic in nature, vagal preganglionic fibers exert their effects on the inhibitory myenteric plexus neurons via both nicotinic and muscarinic receptors. Excitatory and inhibitory neural activity influences LES resting tone by affecting the myogenic tone. Excitatory myenteric plexus neurons release Ach and substance P to increase LES pressure. The inhibitory myenteric plexus neurons decrease LES pressure with NO being the most dominant inhibitory neurotransmitter that is also involved in swallow-induced LES relaxation. The sympathetic efferents innervating the LES originate in spinal segments T6–T10. The splacnhnic preganglionic fibers are cholinergic and target the celiac ganglia to activate noradrenergic postganglionic neurons that either directly innervate LES smooth muscle or synapse onto the enteric motor neurons.Lysophosphatidic acid (LPA) is a bioactive lipid mediator that is released by activated platelets and is constitutively present in serum. It has been identified to be a potent phospholipid messenger with a variety of biological actions, which include cell proliferation, survival and migration, wound healing, platelet aggregation, vascular remodeling, neurite retraction, differentiation inhibition/reversal, membrane depolarization, formation of focal adhesion and stress fibers, blood pressure regulation and smooth muscle contraction.LPA exhibits its functions mainly through binding to its specific G protein-coupled receptors (GPCRs). Currently, there are six GPCRs identified as specific receptors for LPA that are referred to as LPA1-6. LPA1-3were identified as members of the endothelial differentiation gene (Edg) subfamily of GPCRS, as they share a high homology with each other. By contrast, LPA4-6belong to the non-Edg family.It is demonstrated that esophageal motor disorders such as nutcracker esophagus, achalasia and diffuse esophageal spasm showed abnormalities of the lower esophageal sphincter or/and esophageal muscles. Recent studies have demonstrated that the regulatory mechanism of the LES involves various receptors, signal transduction pathways, and neurotransmitter, and play a role in the regulation of the LES at home and abroad.Reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting, measurement of muscle tension in vitro, and electrical field stimulation (EFS) were used to identify expression and function of the LPA receptors in the human LES. The present study investigated the role that the LPA receptors play in modulating human LES function. So that we can demonstrate the regulatory mechanism of the LES much more properly, and provide theoretical bases for the clinical treatment of esophageal motility disorders. PartⅠ Expression of LPA receptors in the human lower esophagealsphincterObjective: LPA receptor is a member of the G protein-coupled receptor family. In the present study, we identified the expression of mRNA and protein of LPA receptors in four muscle strips including sling fibers, clasp fibers, circular muscle strips of the esophagus and stomach by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR), western blotting.Methods: The muscle strips were selected from15patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from January2012to July2012for the study. There were9male and6female patients, with an average age of about64years. Fresh esophagogastric junction specimens were collected in the operating room, in which the sling fibers, clasp fibers and circular muscle strips of esophagus and stomach were separated. Total RNA was extracted. After the identification of its purity and integrity, reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers designed specifically to match the mRNA of LPA receptors. After mRNA expression in four kinds of muscle strips were determined, the relative expression level measued by real-time quantitative PCR. Total proteins were extracted from organizations, and adjusted to the same protein concentration. After electrophoretic separation of the various subtypes of LPA receptor they were transferred onto a polyvinylidene difluoride, respectively. At last the detection of the protein expression was operated using various LPA receptors’ polyclonal antibody. Gel Pro software was used to measured the integrated optical density (IOD) of each receptor bands.Results: The value of A260/280of total RNA was between1.8and2.0after ultraviolet spectrophotometry. Transcripts for LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R were identified in the various muscle strips. The length of the amplification product was consistent with the expected size. Significant differences were demonstrated when comparing the expression of different LPA receptors’ mRNA in the same muscle strips (F=61.034, P= 0.000). The rank order of the extent of expression was LPA1R>LPA6R>LPA4R=LPA2R=LPA5R=LPA3R. However, there was no significant difference in mRNA expression of LPA receptors between the four muscle strips.(F=0.201, P=0.895). Protein expression of six LPA receptor subtypes were identified too, they were LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R, their molecular was39KD,40KD,42KD,42KD,41KD, and39KD, respectively. There was a significant difference in relative expression level for different LPA receptors in the same muscle strip (F=1224.659, P=0.000). The rank order of relative expression level was the same as the result of the RT-PCR. There was no significant difference in relative expression level between the four muscle strips.(F=0.039, P=0.990).Conclusion: There are six kinds of LPA receptor subtypes in human LES, namely LPA1R, LPA2R, LPA3R, LPA4R, LPA5R and LPA6R. Their relative expression levels is LPA1R> LPA6R> LPA4R=LPA2R=LPA5R=LPA3R. They may play an important role in the regulation of human LES function.Part Ⅱ The role of LPA receptors in modulating human lower esophageal sphincterObjective: To identify the effects of selective and non-selective LPA receptors agonists on the sling fibers and clasp fibers of the human LES，and explore the regulatory mechanisms of LPA receptors in human LES contraction and relaxation.Methods: The muscle strips were selected from30patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from July2012to March2013for the study. There were19male and11female patients, with an average age of about62years. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens were fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture. The specimen was cutted from the greater curvature of the stomach. Sharp dissection of its cardia mucosa and submucosa of the lower esophagus were performed and then the thickening of the muscle layer at the esophagogastric junction was found. The sling fibers could be identified in the gastric cardia, adjacent to the lesser curvature of the stomach. The clasp fibers could be identified adjacent to the lesser curvature of the stomach. The sling fibers and clasp fibers were dissociated and prepared into (2~4) mm×(8~12) mm muscle strips. Both ends of the muscle strips were fastened with silk, and placed in a10ml bath containing Krebs liquid, maintained a constant temperature of37℃and persisted through the gas containing5%CO2and95%O2. The upper of muscles and JZ101type muscle tension transducer were fastened together, Medlab signal acquisition applications recorded the changes in individual muscle tension. The muscle strips were pulled slowly and make the tension reaching200mg, this tension of the muscle strip is the initial length L0, Then the muscle strips were pulled slowly and repeatedly, each pulled about25%of the initial length, until the muscle strips were stretched to200%of the initial length which is as the optimum initial length. The optimal initial length of the muscle were stabilize about40minutes, then non-selective LPA receptor agonists were added into thermostatic bath to activate each LPA receptor subtypes, which was a cumulative manner from10-9to10-3mol/L. Each concentrations of the drug being added after the reaction of the previous concentration reached a maximum. The cumulative administration concentration-response dose-response curves were established due to the above results. And the maximum effect after dosing and its corresponding concentration were calculated. When observing the effects of antagonists, the concentration of the antagonist was found with a concentration of agonist-induced muscle maximal effect is the same. The administration of selective LPA receptors agonist and selective LPA receptors antagonist were in the same method. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error. Results:1Effect of non-selective LPA receptor agonist and antagonist on the human LES.The non-selective dopamine receptor agonist LPA induced the contraction of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). The response induced by non-selective dopamine receptor agonist Tetradecyl-phosphonate at the concentration of10-5mol/L, was inhibited completely by non-selective dopamine receptor antagonist Tetradecyl-phosphonate (10-5mol/L).2Effect of selective LPA1and LPA2receptor agonist on the human LES.The selective LPA1and LPA2receptor agonist L-α-Lysophosphatidic acid induced a concentration-dependent contractile response of the clasp and sling fibers of the human LES at the concentration of (10-6,10-5,10-4mol/L). There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/L. The maximum contraction percentage of clasp fibers was (25.3±1.1)％. The maximum contraction percentage of sling fibers was (23.8±0.9)％. There was no significant difference (P＞0.05).3Effect of selective LPA3receptor agonist on the human LES.The selective LPA3receptor agonist OMPT induced contraction of the human LES at the concentration of (10-6,10-5,10-4mol/L), which was in a concentration-dependent manner too. There was no significant difference in contraction between the clasp and sling fibers (P＞0.05). The optimal concentration leading to maximum contraction percentage was10-5mol/l. The maximum contraction of clasp fibers was (7.2±0.4)％. The maximum relaxation of sling fibers was (7.8±0.6)％. There was no significant difference (P＞0.05).Conclusions:1The non-selective Lysophosphatidic acid receptor agonist can induce the contraction of the human LES. The reaction of the human LES can be inhibited completely by non-selective Lysophosphatidic acid receptor antagonist. This study indicates that LPA regulate the lower esophageal sphincter is through its receptor.2The selective LPA1and LPA2receptor agonist induces a concentration-dependent contractile response. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA1or LPA2, or LPA1and LPA2receptor are involved in the contractile response of the human LES.3The selective LPA3receptor agonist induces contraction of the human LES, which is in a concentration-dependent manner too. The optimal concentration leading to maximum contraction percentage is10-5mol/l. There is no significant difference in contraction between the clasp and sling fibers. This study indicates that LPA3receptor is involved in the contraction of the human LES.PartⅢ The contribution of LPA receptors in the response of human lower esophageal sphincter under the electical field stimulationObjective: To identify the effect that LPA receptor subtypes play the role in the clasp fibers and sling fibers of the human lower esophageal sphincter (LES) under the electical field stimulation (EFS), and investigate the role of LPA receptor subtypes in vagal pathways that regulating human LES function.Methods: The muscle strips were selected from20patients who underwent esphagectomy for mid-third esophageal carcinoma at the Fourth Hospital of Hebei Medical University from March2013to December2013for the study. There were19male and11female patients, with an average age of about62years. The clasp fibers and sling fibers were prepared using the similar methods that were described previously. The fresh esophagogastric junction was collected in the operating room and then immediately placed in4℃Krebs, After washed specimens is fixed within the wax disc which containing Krebs liquid, and maintain a gas containing5%CO2and95%O2mixture continued through. The upper of muscles and JZ101type muscle tension transducer were fastened together, while the lower end and an L-shaped bracket with platinum electrodes were fastened together. Medlab signal acquisition applications were used to record changes in individual muscle tension. All the muscles were ensured in the middle of the platinum electrode, which was more than3mm. Physiology and Pharmacology multi-purpose instrument was used for electrical field stimulation. The optimum initial length was ensured using the similar methods that were described previously. Electrical stimulation was conducted according to the frequency from small to big and the maximum effect after stimulation was calculated. Then the muscle strip was stimulated again after20min of administration of selective LPA1and LPA3receptor antagonist Ki16425at the concentration of10-5mol/L. The responses in all of the experiments were quantified based upon a percentage of the baseline value of muscle strip tone relative to the nadir of the response. The data were expressed as means±standard error.Results:1The EFS induced a frequency-dependent relaxation in clasp fibers of The LES. When the end of the relaxation response every time, there was a quick rebound contraction which was in the frequency-dependent manner too. The optimal frequency resulting in maximum relaxation percentage was64Hz. The maximum relaxation was (17.8±0.7)％.The EFS induced a frequency-dependent contraction in sling fibers. The optimal frequency resulting in maximum contraction percentage was128Hz. The maximum contraction was (17.0±0.8)％.2Effect of the selective LPA1and LPA3receptor antagonist Ki16425on the clasp and sling fibers of the human LES under the EFS: The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced by the EFS (P＞0.05). The selective LPA1and LPA3receptor antagonist (10-5mol/L) produced no significant change in the frequency-dependent contraction in sling fibers of the human LES induced by the EFS (P＞0.05). Conclusions:1The EFS induce frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter. The optimal frequency resulting in maximum relaxation is64Hz. EFS induces frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter. The optimal frequency leading to maximum contraction is128Hz.2The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent relaxation in clasp fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that LPA1and LPA3receptors are not involved in the response of clasp fibers of the human lower esophageal sphincter induced by the EFS.3The selective LPA1and LPA3receptor antagonist produce no significant change in the frequency-dependent contraction in the sling fibers of the human lower esophageal sphincter induced by the EFS. This study indicates that the LPA1and LPA3receptors are not involved in the response of sling fibers of the human lower esophageal sphincter induced by the EFS. |
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