| The lower esophageal sphincter (LES) is a thickened region of thecircular muscle layer located at the gastroesophageal junction in human,extending over an axial distance of2–3cm. In1979, Liebermann-Mefferproposed that musculature of equivalent of the LES consists of clasp fibers atthe lesser curvature and sling fibers at the greater curvature. These two musclefibers and the crural diaphragm are in a state of constant or tonic contraction,which is due to both myogenic and neurogenic elements of the LES, and forma high-pressure zone at the gastroesophageal junction to prevent reflux of thegastric contents into the esophagus; on the other hand, swallow as well asesophageal distension induced the transient LES relaxation (TLESR), whichpermits the passage of food from the esophagus into the stomach or to vomitand hiccup, by which induced the reflux of the gastric contents into theesophagus.The functional regulation of contraction and relaxation of the LES iscompleted by several hormone, neurotransmitter and spontaneous myogenicfactors under the control of the the central nervous system. The vagal efferentfibers synapse with a huge number of enteric motor neurons (EMN), which arelocated in the myenteric plexus (MP), and the can form both inhibitory andexcitatory pathways by the synapse. The preganglionic neurons that form theinhibitory and the excitatory pathway are cholinergic. Furthermore, theneurotransmission of the excitatory postganglionic neurons is cholinergic,whereas the inhibitory postganglionic neurons are nonadrenergicnoncholinergic (NANC). Both the inhibitory and the excitatory vagalpathways exert tonic effects on the LES. The activation of the vagal excitatorypathway can generate contraction of the LES, whereas the activation of thevagal inhibitory pathway can generate relaxation of the LES. Dopamine (DA), as a predominant catecholamine neurotransmittercontrols, a variety of functions, including locomotor activity, cognition,emotion, endocrine regulation and gastrointestinal motility. All of thesefunctions are mediated by DA receptors, which are important members of theseven transmembrane domain G protein-coupled receptor family. DAR hasbroad expression patterns in both central nervous system and peripherynervous system. By now, five distinct DA receptor subtypes (D1R-D5R) havebeen found. On the basis of their structural, pharmacological, and biochemicalproperties, these receptors were classified as either D1-like dopaminereceptors (D1R and D5R) or D2-class dopamine receptors (D2R, D3R, andD4R). Recent experiments have suggested that DA receptors are widelyexpressed in animal gastrointestinal tract.Various esophageal motility disorders, such as achalasia, diffuseesophageal spasm and nutcracker esophagus, are all associated with motordisorders of the LES. Recent studies have demonstrated that the regulatorymechanism of the LES involves various receptors, neurotransmitter, and signaltransduction pathways. CCK receptors, and Muscarinic receptors have beendemonstrated to play a role in the regulation of the LES.Reverse transcription-polymerase chain reaction (RT-PCR), westernblotting, measurement of muscle tension in vitro, and electrical fieldstimulation (EFS) were used to identify expression and function of the DAreceptors in the human LES. The present study investigated the role that theDA receptors play in modulating human LES function. So that we candemonstrate the regulatory mechanism of the LES much more properly, andprovide theoretical bases for the clinical treatment of esophageal motilitydisorders.Partâ… Expression of dopamine receptors in human lower esophagealsphincterObjective: Dopamine (DA) receptors as a member of the Gprotein-coupled receptor family, mediate all of the physiological functions of the catecholaminergic neurotransmitter dopamine. In the present study, weidentified the expression of mRNA and protein of DA receptors in four musclestrips including sling fibers, clasp fibers, circular muscle strips of esophagusand gastric with the use of reverse transcription-polymerase chain reaction(RT-PCR) and western blot, to investigated the role that the DA receptors playin modulating human LES function.Methods: Thirty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between December2007and October2008were selected in thisstudy. There were23male and7female patients, with a mean age of64years.Each specimen including part of the gastric fundus, the gastroesophagealjunction, and the esophageal body, was resected en bloc in the operating room.After the mucosa and submucosa were removed by sharp dissection, the slingand clasp fibers of LES, and circular muscle strips of esophagus and gastricwere obtained from various regions of the gastroesophageal junction andadjacent structures. The dissected muscle strips were frozen in liquid nitrogenand stored at-80°C for subsequent RNA and protein extraction. Total RNAwas extracted by acid guanidinium thiocyanate-phenolchloroform extraction.After the identification of its purity and integrity with the use of ultravioletspectrophotometer and1%agarose gels, reverse transcription-polymerasechain reaction (RT-PCR) was performed using primers designed specificallyto match the DA receptors' mRNA, to investigate the mRNA expression ofDA receptors. The unit of integrated optical density (IOD) of the amplifiedproducts was calculated with Gel-Pro software. The expression of DAreceptor mRNA was expressed by the ratio of IOD value of DA receptor bandto β-actin band. The integral membrane protein receptors, which wereextracted from muscle tissue, were quantitated and adjusted to the identicalconcentration, the different DA receptors were separated by electrophoresis.At last the detection of the protein expression was operated using different DAreceptors' polyclonal antibody after the trarsmembrane. The IOD value wasalso calculated with the Gel-Pro software. Results: The value of A260/280of total RNA was between1.6and1.8after ultraviolet spectrophotometry. The width and brightness of28S bandwere double than18S band in1%agarose gels. The band of β-actin mRNAwas uniformly838bp. Transcripts for D1R, D2R, and D5R were identified inthe four muscle strips. D3R and D4R mRNA were not identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent DA receptors' mRNA in the same muscle strips (F=669.00, P=0.00). The rank order of the extent of expression was D1R>D5R> D2R.However, there was no significant difference in mRNA expression of DAreceptors between the four muscle strips.(F=0.18, P=0.90). Proteinexpression of three DA receptor subtypes were identified, they were D1R, D2Rand D5R, with respective molecular,51KD,51KD and53KD. D3R and D4Rprotein expression were not identified in the four muscle strips. There was asignificant difference in IOD values for different DA receptors in the samemuscle strip (F=84.53, P=0.00). The rank order of the value was the sameas the result of the RT-PCR. There was no significant difference in IOD valuesbetween the four muscle strips.(F=0.10, P=0.96).Conclusion: D1R,D2R,D5R can be detected in the human LES, the rankorder of the extent of expression is D1R>D5R> D2R, and probably contributeto LES function. D3R and D4R are not expressed, and probably do notcontribute to LES function in humans.Partâ…¡ The role of dopamine receptors in modulating human loweresophageal sphincterObjective: To identify the effect that non-selective dopamine receptoragonist, antagonist and selective dopamine receptor agonist have played inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES), and investigate the role of dopamine receptor subtypes inmodulating contraction and relaxation of the LES.Methods: Thirty patients who underwent esophago-gastrectomy for mid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between August2010and March2011were selected in this study.There were17male and13female patients, with a mean age of62years. Eachspecimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. In the laboratory, the surgical specimen wasopened along the long axis of the esophagus and the greater curvature of thestomach. It was washed with37°C Krebs solution which had previously beenbubbled with95%O2and50%CO2. The specimen was then pinned on a waxplate with the mucosal surface facing up to maintain its approximate in situdimensions. Then both the mucosa and submucosa were removed by sharpdissection. The LES was recognized as a thickened band of circular muscle atthe gastroesophageal junction. The sling and clasp fibers could be identified asthickened bands of circular oriented smooth muscle in the gastric cardia,adjacent to the greater and lesser curvature of the stomach, respectively.Transversely oriented strips measuring2mm×10mm were prepared from thesling and clasp fibers so that the long axis of the muscle strip paralleled thelong axis of smooth muscle cells constituting the circular muscle layer. Eachend of the muscle strips were attached with silk suture, and one of the end wastied to to an isometric force transducer supported on a rack-and-pinion clampto facilitate an accurate length adjustment of the muscle strips. Tension wasrecorded the software of MedLab6.0. The strips were incubated1h in the10-ml jacketed tissue baths filled with Krebs solution maintained at37°C andbubbled continuously with95%O2-5%CO2. Each muscle strip was stretchedslightly and rapidly until200mg of force was generated. This was taken asthe initial length (L0). The muscle strips were then sequentially stretched to200%of the L0, at the increments of25%of the L0each time. This was takenas the most suitable initial length. The muscle strips were equilibrated for40min in in the10-ml jacketed tissue baths filled with Krebs solution maintainedat37°C and bubbled continuously with95%O2-5%CO2. The administration of non-selective dopamine receptor agonist Dopamine hydrochloride (DA)was a cumulative manner from10-9to10-3mol/L, each administration beingdone when the response of the previous concentration reached a maximum.The concentration of the non-selective dopamine receptor antagonistFlupenthixol dihydrochloride was identical with optimal concentration leadingto maximum response. All of the drug concentrations are final concentrationsin the tissue bath. The administration of selective D-1like receptor agonist(±)-SKF-3893and selective D2R agonist (-)-Quinpirole hydrochloride were inthe same way. The responses in all of the experiments were quantified basedupon a percentage of the baseline value of muscle strip tone relative to thenadir of the response. The data were expressed as means±standard error.Results:1Effect of non-selective dopamine receptor agonist and antagonist on thehuman LESThe non-selective dopamine receptor agonist DA induced the contractionof the clasp and sling fibers of the human LES at the concentration of (10-7,10-6,10-5,10-4,10-3mol/L), while relaxation induced at the concentration of(10-5,10-4,10-3mol/L). The response induced by non-selective dopaminereceptor agonist Flupenthixol dihydrochloride at the concentration of10-4mol/L, was inhibited completely by non-selective dopamine receptorantagonist Flupenthixol dihydrochloride (10-4mol/L).2Effect of selective D-1like receptor agonist on the human LESThe selective D-1like receptor agonist (±)-SKF-3893induced aconcentration-dependent contractile response of the clasp and sling fibers ofthe human LES at the concentration of (10-7,10-6,10-5,10-4,10-3mol/L). Therewas no significant difference in contraction between the sling fibers and claspfibers (F=0.401, P=0.808). The optimal concentration leading to maximumcontraction was10-4mol/L. The maximum contraction of clasp fibers was(15.8±2.2)ï¼…. The maximum contraction of sling fibers was (17.8±2.1)ï¼….There was no significant difference (F=0.015, P=0.903).3Effect of selective D2R agonist on the human LES The selective D2R agonist (-)-Quinpirole hydrochloride inducedrelaxation of the human LES at the concentration of (10-5,10-4,10-3mol/L),which was also in a concentration-dependent manner. There was no significantdifference in relaxation between the sling fibers and clasp fibers (F=1.258,P=0.292). The optimal concentration leading to maximum relaxation was10-3mol/l. The maximum relaxation of clasp fibers was (12.0±1.6)ï¼…. Themaximum relaxation of sling fibers was (10.8±2.1)ï¼…. There was nosignificant difference (F=0.131, P=0.721).Conclusion:1The non-selective dopamine receptor agonist can induce the contraction ofthe human LES at a low concentration, relaxation occurs with the rise of theconcentration. The reaction of the human LES can be inhibited completely bynon-selective dopamine receptor antagonist. This study indicates that DARinvolves the reaction of LES to DA.2The selective D-1-like receptor agonist induces a concentration-dependentcontractile response. The optimal concentration leading to maximumcontraction is10-4mol/l. There is no significant difference in contractionbetween the sling fibers and clasp fibers. This study indicates that the D-1-likereceptor is involved in the contractile response of the human LES.3The selective D2R agonist induces relaxation of the human LES, which isalso in a concentration-dependent manner. The optimal concentration leadingto maximum relaxation is10-3mol/l. There is no significant difference inrelaxation between the sling fibers and clasp fibers. This study indicates thatD2R is involved in the relaxation of the human LES.Partâ…¢ The contribution of dopamine receptors in the response ofhuman lower esophageal sphincter under the electical field stimulationObjective: To identify the effect that dopamine receptor subtypes play inregulating the sling fibers and clasp fibers of the human lower esophagealsphincter (LES) under the electical field stimulation (EFS), and investigate therole of dopamine receptor subtypes in vagal pathways that modulating human LES function.Methods: Twenty patients who underwent esophago-gastrectomy formid-third esophageal carcinoma at the Fourth Hospital, Hebei MedicalUniversity between March2011and November2011were selected in thisstudy. There were14male and6female patients, with a mean age of58years.Each specimen was resected en bloc in the operating room, and after surgicalexcision, it was placed immediately in ice-cold Krebs solution. Specimenswere not included in this study if any segment required for study containedmacroscopically visible tumour. The sling and clasp muscle strips wereprepared using similar methods to those we have described previously. Eachend of the muscle strips were attached with silk suture. The muscle strip wastied at one end to the electrode holder and at the other end was tied to to anisometric force transducer supported on a rack-and-pinion clamp to facilitatean accurate length adjustment of the muscle strips. Tension was recorded thesoftware of MedLab6.0. The muscle strips were mounted through concentricplatinum electrodes, makesure the distance between the end and electrode wasmore than3mm. The electrodes were connected to the output of thestimulator that delivered single pulse square wave, trains of0.5ms,50-Vsquare wave pulses at1～512Hz. Before the study, each muscle strip wasgently stretched to the the most suitable initial length, just like what we haveshown previously. The responses of muscle strips to EFS were sequentiallyassessed over a frequency range of1～512Hz, with5min between eachfrequency under control conditions. Then the muscle strip was stimulatedagain after20min of administration of selective D-1like receptor antagonistSCH23390at the concentration of10-4mol/L. The administration of selectiveD2R antagonist Spiperone hydrochloride at the concentration of10-3mol/Lwas in the same way. The responses in all of the experiments were quantifiedbased upon a percentage of the baseline value of muscle strip tone relative tothe nadir of the response. The data were expressed as means±standard error.Results:1Effect of the EFS on the the clasp and sling fibers of the human LES The EFS induced a frequency-dependent relaxation in clasp fibers. Aftereach of the stimulation, there was a quick rebound contraction which was alsoin the frequency-dependent manner. The optimal frequency resulting inmaximum relaxation was64Hz. The EFS induced a frequency-dependentcontraction in sling fibers. The optimal frequency resulting in maximumcontraction was128Hz. The maximum contraction was (13.6±1.8)ï¼….2Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the clasp fibers of thehuman LES under the EFSThe selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent relaxation in claspfibers of the human LES induced by the EFS (F=1.142, P=0.342). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS (F=0.359, P=0.837).3Effect of the selective D-1like receptor antagonist SCH23390and theselective D2R antagonist Spiperone hydrochloride on the sling fibers of thehuman LES under the EFSThe selective D-1like dopamine receptor antagonist (10-4mol/L)produced no significant change in the frequency-dependent contraction insling fibers of the human LES induced by the EFS (F=0.354, P=0.840). Theselective D2R antagonist (10-3mol/L) produced no significant change in thefrequency-dependent contraction in sling fibers of the human LES induced bythe EFS (F=0.651, P=0.627).Conclusion:1The EFS induces frequency-dependent relaxation in clasp fibers. Theoptimal frequency resulting in maximum relaxation is64Hz. EFS inducesfrequency-dependent contraction in the sling fibers. The optimal frequencyleading to maximum contraction is128Hz.2Both of the selective D-1like dopamine receptor antagonist and theselective D2R antagonist produces no significant change in the frequency-dependent relaxation in clasp fibers of the human LES induced bythe EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of clasp fiers of the human LES induced by the EFS.3Both of the selective D-1like dopamine receptor antagonist and theselective D2R antagonist produces no significant change in thefrequency-dependent contraction in the sling fibers of the human LES inducedby the EFS. This study indicates that the D-1like receptor and D2R are notinvolved in the response of sling fiers of the human LES induced by the EFS.
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