β-adrenoceptors Expression And Function In Human Lower Esophageal Sphincter

The lower esophageal sphincter （LES） at the gastroesophageal junction isa special thickened circular muscle layer about2-3cm in human. In1979,Liebermann-Meffer proposed firstly that the huanm lower esophagealsphincter consists of sling fibers at the greater curvature and clasp fibers at thelesser curvature. The clasp fibers, the sling fibers and the crural diaphragmregulated with the mediation of the nervers, body fluids and other factors,maintain a continued contraction, which forms a high pressure band atesophagogastric and constitute the physilolgical antireflux barrier to preventthe reflux of the gastric contents into the esophagus.The adjustment mechanism of contraction and relaxation of the LES isrugulated by several neurotransmitters, hormones and spontaneous myogenicfactors in the control of the central nervous system. The output nerve endingof the vagus nerve and the intestinal motor neurons of the lower esophagealsphincter constitute the inhibitory and excitatory movement pathway. Theexcitatory movement pathway consists of preganglionic cholinergic sportsneurons and postganglionic cholinergic motor neurons and the inhibitorymovement pathway consists of preganglionic cholinergic motor neurons andpostganglionic non-cholinergic non-adrenergic neurons. Activation ofexcitatory or inhibitory neural pathway leads to the release of variety ofneurotransmitte of cholinergic postganglionic neurons or intestinal neurons,which could adjust the contraction or relaxation of the lower esophagealsphincter. Now it is obvious that the activation of the excitatoryneurotransmitter pathway could release acetylcholine and substance P, whichlead to the contraction of the lower esophageal sphincter. The activation of theinhibitory neurotransmitter pathway could release nitric oxide and vasoactiveintestinal peptide, which lead to the relaxation of the lower esophageal sphincter.It is demonstrated that esophageal motor disorders such as diffuseesophageal spasm, achalasia and nutcracker esophagus showed abnormalitiesof the lower esophageal sphincter. Therefore, the study of the regulationmechanism of the lower esophageal sphincter is important for the diagnosisand treatment of esophageal motility disorders. The studies showed that theregulatory mechanism of the LES involves of Nitric oxide, acetylcholine,vasoactive intestinal peptide, their receptors snd signal transduction pathways.β-adrenergic receptor is a member of the G protein-coupled receptorfamily. It has been demonstrated that there are three receptor subtypesincluding β₁-AR, β₂-AR, β₃-AR, which are widely present in themammalian central and peripheral nervous system, such as brain,cardiovascular system and gastrointestinal tract, and are importantphysiological modulatorBy western blotting, reverse transcription-polymerase chain reaction （RT-PCR）, measurement of muscle tension in vitro and electrical field stimulation（EFS），the purpose of this thesis is to investigateβ-AR expression andfunction of the lower esophageal sphincter and to investigate the β-AR inhuman esophageal sphincter adjustment mechanism, which could furtherimprove the human lower esophageal sphincter regulating mechanisms andprovide a theoretical basis for the treatment of esophageal motility disorders.Part Ⅰ Expression of β-adrenergic receptor in the human loweresophageal sphincterObjective:β-adrenergic receptor is a member of the G protein-coupledreceptor family. In the present study, we identified the expression of mRNAand protein of β-AR receptors in four muscle strips including sling fibers,clasp fibers, circular muscle strips of the esophagus and stomach by westernblotting, reverse transcription-polymerase chain reaction （RT-PCR）.Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in the Department of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from January2011to October2011. There were21males and9females, with an average age of64years （ranged55to68）. In the laboratory,fresh esophagogastric junction specimens collected in the operating room wereprepared into the sling fibers, clasp fibers and circular muscle strips ofesophagus and stomach. Total RNA was extracted. Reversetranscription-polymerase chain reaction （RT-PCR） was performed usingprimers designed specifically to match the β-AR mRNA. PCR productswere determined by using the Gel-Pro gel imaging analysis system.Densitometry for bands on PCR products was determined by imaging software.The relative expression level of each gene was normalized by the value ofβ-actin. Total proteins were extracted from muscle strips by using proteinextraction kit. Proteins were adjusted to the same concentration. The threesubtypes were separated by electrophoresis and transferred onto apolyvinylidene difluoride （PVDF）. The detection of the protein expressionwas operated using different β-AR polyclonal antibody. The membrane wasdetected by infrared fluorescence imaging instrument. The relative expressionlevel of each protein was normalized by the value of β-actin..The value of A260/280of total RNA was between1.8and2.0afterultraviolet spectrophotometry. The band of β-actin mRNA was uniformly540bp. Transcripts for β₁-AR, β₂-AR, and β₃-AR were identified in the fourmuscle strips. The PCR product was consistent with the expected size.Significant differences were demonstrated when comparing the expression ofdifferent β-AR mRNA in the same muscle strips （F=4100, P=0.00）. Therank order of the extent of expression was β₃-AR> β₁-AR> β₂-AR.However, there was no significant difference in mRNA expression of β-ARbetween the four muscle strips（F=0.78, P=0.30）. β₁-AR, β₂-AR, andβ3-AR protein expression were identified. There was a significant difference inIOD values for different β-AR in the same muscle strip （F=39, P=0.00）.The rank order of the value was the same as the result of the RT-PCR. Therewas no significant difference in IOD values between the four muscle strips。 Conclusion: β₁-AR, β₂-AR,β₃-AR can be detected in the humanLES, the extent of expression in secending order is β₃-AR>β₁-AR>β₂-AR,and probably contribut to LES function.Part Ⅱ The role of β-adrenoceptors in modulating human loweresophageal sphincterObjective： To study the effects of selectinve and nonselective β-adrenoceptors agonists and antagonists on the LES muscle strips，andinvestigate the role of β-adrenoceptors subtypes in adjusting contraction andrelaxation of the LES.Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from October2011to March2012. There were20males and10females, with an average age of63years. In the laboratory, freshesophagogastric junction specimens collected in the operating room wereimmediately placed in the4℃Krebs. After washed with37℃Krebssolution, specimens were pined on a wax plate containing TBS, withcontinuous mixed gas of95%O₂and5%CO₂. The mucosa and submucosawere then gently removed by sharp dissection. The gastric sling and claspfibers could be identified as thickened bands of circular oriented smoothmuscle in the gastric cardia, adjacent to the greater and lesser curvature of thestomach, respectively. The sling and clasp muscle strips were prepared usingthe method that we have described previously. The sling fibers, clasp fiberswere separated and prepared into （2⁴） mm×（8¹2） mm muscle strips.The ends of the muscle strips were tied with silk, placed in a10ml bathcontaining Krebs solution maintained37℃，with continuous mixed gas of95%O₂and5%CO₂. The upper end of the muscle strips was fixed with JZ101muscle tension transducer and the changes of the muscle strips tension wasrecord by the Medlab signal acquisition system. The muscle strips were stretchslowly and make the tension maintaining at200mg, and the length of the muscle strips at that tension is the initial length L0. The muscle strips werestretch slowly and repeatedly, with each about25%of the initial length, untilthe muscle strips were stretched to200%of the initial length which is as theoptimum initial length.The muscle strips with the optimum initial were stabilized for30minutes, then the non-selective agonists of the β-adrenoceptors subtypewere added into the bath with a cumulative manner from10^-9 to10^-3 mol/Lrespectively. Successive concentrations of the agonists were not added untilthe response of the previous concentration stabilized. Ten minutes betweenadditions were allowed for lack of effect. Based on the above results, we drawthe cumulative administration concentration-response dose-response curves.The antagonist was added before adding the agonist for observed the effect ofthe antagonist and the concentration of the antagonist is same to theconcentration of the agonist that induced the muscle strips to produce themaximum effect. The administration of selectiveβ-adrenoceptors agonist andselectiveβ-adrenoceptors antagonist were in the same way. The responses inall of the experiments were quantified based upon a percentage of the baselinevalue of muscle strip tone relative to the nadir of the response. The data wasexpressed as means±standard error.Results:1Isoproterenol, a non-selective β-adrenoceptor agonist, could inducerelaxation effect of the lower esophageal sphincter muscle strips at theconcentration of10^-4mol/L. Propranolol, non-selective β-adrenoceptorsantagonists was no effect on the relaxation effect induced by Isoproterenol，butwhich could be blocked by L748337, a selective β-adrenoceptor agonist.2Dobutamine, a selectiveβ₁-adrenoceptor agonist, and salbutamol, aselective β₂-adrenoceptor agonist, had no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,could induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect at the concentration from10^-9to10^-3mol/L. The optimal concentration leading to maximum contraction was 10^-4mol/L. The maximum relaxation of the LES muscle strips was（80±0.6）％. L748337, a selectiveβ₃-adrenoceptor antagonist could block theeffect of BRL37344.Conclusion:1soproterenol, a non-selective β-adrenoceptor, induces relaxationeffect of the lower esophageal sphincter muscle strips at the concentration of10^-4mol/L. Propranolol, non-selective β-adrenoceptors antagonists has noeffect on the relaxation effect induced by Isoproterenol，but can be blocked byL748337, a selective β-adrenoceptor agonist. It implys that the β3-adrenoceptor other thanβ₁-and β₂-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.2Dobutamine, a selectiveβ₁-adrenoceptor agonist, and salbutamol,and a selectiveβ₂-adrenoceptor agonist, has no effect on the lower esophagealsphincter muscle strips. BRL37344, a selective β3-adrenoceptor agonist,induce the lower esophageal sphincter muscle strips to produceconcentration-dependent relaxation effect. L748337, a selective β3-adrenoceptor antagonist could block the effect of BRL37344. The β3-adrenoceptor other than β₁-and β₂-adrenoceptors may be involved in theregulation of the lower esophageal sphincter.PartⅢ The contribution of β-adrenoceptor in the response of humanlower esophageal sphincter under the electical field stimulationObjective: To identify the effect that the β-adrenoceptor antagnostsplay in regulating human lower esophageal sphincter （LES） under the electicalfield stimulation （EFS）, and investigate the role of β-adrenoceptos inmodulating human LES function.Methods: The muscle strips were collected from30patients whounderwent esphagectomy for mid-third esophageal carcinoma in theDepartment of Thoracic Surgery, the Fourth Hospital, Hebei MedicalUniversity from March2012to December2012. There were25males and5females, with an average age of60years. The LES muscle strips were prepared using similar methods to that we have described previously. Themuscle strips were placed in a10ml bath containing Krebs solution maintained37℃，with continuous mixed gas of95%O₂and5%CO₂. The upper end ofthe muscle strips were fixed with muscle tension transducer and the lower endwas connected to the L-shaped bracket with a platinum electrode, with usingof the Medlab signal acquisition to record muscle strip tension changes. Themuscle strips were stretched to optimum initial length using similar methodsto that we have described previously and had a warm bath for1hour in Krebssolution with continuous mixed gas of95%O₂and5%CO₂.EFS stimulation parameters: single-pulse square wave, pulse width5ms,voltage50V, frequency1-512Hz. After added atropine, the muscle strips issubjected to electrical stimulation according to the frequency from small to bigand the maximum effect to EFS was assessed. The muscle strip was stimulatedagain after20min of administration of Tetrodotoxin, Nebivolol （a selectiveβ1-adrenoceptor antagonists）, ICI118551（a selective β₂-adrenoceptorantagonist） and L-748337（a selective β₃-adrenoceptor antagonist）. Theresponses in all of the experiments were quantified based upon a percentage ofthe baseline value of muscle strip tone relative to the nadir of the response.The data was expressed as means±standard error.Results:1The EFS could induce the lower esophageal sphincter （clasp fibers andsling fibers） to produce the frequency dependent of the relaxation response,and the maximum diastolic electrical stimulation frequency was64HZ. Themaximum relaxation percentage of the lower esophageal sphincter byEFS-induced was （22.1±0.4）%.2Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration.3Nebivolol and ICI118551had no effect on the relaxation of the LESmuscle strips by EFS-induced. There was significant no difference before and after administration. L-748337could inhibited the the frequency dependenceof the relaxation response of the LES by EFS-induced. There was significantdifference before and after administration.Conclusion:1Tetrodotoxin significantly reduced the frequent-dependent relaxationin the lower esophageal sphincter by EFS-induced. There was significantdifference in relaxation of the LES muscle strips before and afteradministration. It is prompted that the frequency-dependent relaxation oflower esophageal sphincter by EFS-induced is neurogenic in origin.2Nebivolol and ICI118551have no effect on the relaxation of the LESmuscle strips by EFS-induced. The frequency-dependent contraction in thesling fibers of the human LES can be induced by the EFS. The β3-adrenoceptor other thanβ₁-and β₂-adrenoceptor may be involved in thefrequency-dependent relaxation of the human LES induced by the EFS.