神经节内板状末梢是豚鼠食道迷走传入神经末梢的机械敏感性受体

171ActaPhysiologicaSinica,April25,2006,58(2):171-176*Correspondingauthor.Tel:[phone];Email:[e-mail]endingsactasmechanoreceptorsofvagalafferentnerveinguineapigesophagusYANGXia*,LIURanQiluHospital,ShandongUniversity,Jinan250012,ChinaAbstract:Intraganglioniclaminarendings(IGLEs)havebeensupposedtobethemechanoreceptorsinthegutbyelectrophysiologicalrecordingtechniques.ButthespecializedmorphologyofIGLEscouldnotbedisplayedcloselyassociatedwiththisfunctionandthemechanismthatIGLEsactasthemechanotransductionsitesinthegutisnotyetwellunderstood.Inthepresentstudy,weusedstyryldyeFM1-43combinedwithstretchstimulationintheguineapigesophagustotestwhetherIGLEsactedasthemechano-sensitivereceptorsofthevagalafferentnerves.Atthesametime,thespecialstructureofIGLEsdisplayedbyFM1-43wasfurtherconfirmedbyneurobiotinanterogradelabelingtechnique.TofurtherinvestigatethecharacteristicsofIGLEsasmechanosensitivereceptors,differentdrugswereusedtoblockorstimulateIGLEsactivation.OurresultsindicatedthatonlyinthestretchedpreparationcouldFM1-43entertheIGLEsandcompletelydisplaytheirspecializedstructure,whichwasconsistentwiththatshownbyneurobiotin.TheamountofIGLEsshownbystretch-evokedFM1-43stainingwasmuchmorethanthatshownwithoutstretchstimulation[(90.4±9.5)%vs(10.7±2.1)%,P0.05].Ca2+,TTX(0.6µmol/L),atropine(0.6µmol/L),SKF(50µmol/L),andgadolium(100µmol/L)hadnoeffectontheIGLEsactivation.Butforbenzamil(100µmol/L),anepithelialsodiumchannelblocker,activationofIGLEsbystretchstimulationwassignificantlyblocked.ThepotentATPanalogue,α,β-methyleneATP(100µmol/L)couldnotactivateFM1-43stainingwithoutstretch.TheseresultsindicatethatIGLEsaresensitivetomechanicalstimulation.ThiscouldleadtothedeductionthatIGLEsactasthemechanoreceptorsofvagalafferentnerve.IGLEscouldtransmitmechanicalstimulidirectlythroughionchannels,independentofneurotransmitterreleaseandactionpotentialpropagation.Thestretch-sensitivechannelsonIGLEsprobablybelongtotheepithelialsodiumchannelfamilyratherthanvoltage-gatedsodiumionchannels.Furthermore,styryldyeFM1-43isausefulactivity-dependentmarkertodemonstratethestructureandfunctionofIGLEsinguineapigesophagus.Keywords:intraganglioniclaminarendings;mechanotransduction;mechanoreceptors;receptor;FM1-43;anterograde;labeling神经节内板状末梢是豚鼠食道迷走传入神经末梢的机械敏感性受体杨霞*，刘然山东大学齐鲁医院，济南250012摘要：电生理学研究发现迷走传入神经在胃肠道的特有结构——神经节内板状末梢(intraganglioniclaminarendings,IGLEs)具有感受机械刺激的功能，推断其为迷走神经机械敏感性受体。但是电生理学方法不能将IGLEs的特异结构与其感受机械刺激的功能同时显示出来，而且IGLEs作为机械敏感性受体，其传导机械刺激的机制尚不清楚。本研究应用活性依赖性荧光染料FM1-43结合牵拉刺激豚鼠食道显示激活的IGLEs结构，以期观察IGLEs是否对机械刺激敏感。同时用多种药物阻断或促进豚鼠食道IGLEs的激活以探讨IGLEs传导机械刺激的机制。应用神经顺行标记技术以验证FM1-43显示的特异结构是否为IGLEs。结果表明，牵拉刺激结合FM1-43染色显示的结构与神经顺行标记法一致，牵拉刺激组激活的IGLEs数目明显多于未牵拉组[(90.4±9.5)%vs(10.7±2.1)%,P0.05]。IGLEs对牵拉刺激的敏感性，表明IGLEs是迷走传入神经在胃肠道内感受机械刺激的受体。TTX，阿托品和钙离子对牵拉刺激激活IGLEs无明显影响，表明IGLEs对机械刺激的传导不需要神经递质以及动作电位的传导，而是直接通过机械门控离子通道实现的。多种TRP通道阻断剂包括SKF,gadolinium对IGLEs的激活无影响，而上皮钠离子通道阻断剂benzamil可以明显阻断IGLEs的激活，因此推断，IGLEs结构中传导机械刺激的离子通道可能属于上皮钠离子通道家族而非电压门控钠离子通道或TRP通道。ActaPhysiologicaSinica,April25,2006,58(2):171-176172关键词：神经节内板状末梢；机械传导；机械受体；受体；FM1-43；顺行；标记中图分类号：R338Thespecializedafferentvagalterminalendingsintheguthavebeendescribedasintraganglioniclaminarendings(IGLEs).ThemorphologyofIGLEshasbeendescribedbyanterogradelabelingandimmunohistochemistrytech-niquesasspecificflattened,leaf-likestructurecirclingaroundthemyentericganglia.Byusingelectrophysiologi-calrecordingtechniquetofindthe“hotspot”,whichissensitivetovonFreyhairstimulation[1,2],ZagorodnyukandBrookssupposedthatIGLEsactasthetransductionsitesofvagalmechanoreceptorsinguineapigesophagealandstomachtissue.However,thespecializedmorphologyofIGLEscouldnotbedisplayedcloselyassociatedwiththisfunction.Moreover,therearemanyotherionchannelsbesidesIGLEsthatcanmodulatetheactionpotentialsbe-ingrecorded.ThismeansthatelectrophysiologyrecordingisanindirectstudyandthemechanismthatIGLEstrans-mitthemechanicalstimulationinthegutcouldn’tbewellunderstoodbythistechnique.FM1-43isacationicstyryldyethatprovidesfluorescentsignalvisibleunderfluoresceinopticswhenitisinahydro-phobicenvironment[3].TodatetheFMfamilyofdyeshasbeenusedprimarilytoinvestigatemembranetrafficking,astheyaretakenupbyendocytosis[4-6].However,FM1-43couldalsobeusedtodisplaythemechanoreceptorsinver-tebratehaircellsbyanovelmechanismforentryintosen-sorycellsandneurons,namelypermeationthroughmechanosensitive,non-selectiveionchannels[7,8].FM1-43labelingisactivity-dependent,whichmeansthatclosedornonfunctionalchannelsdonotpassFM1-43.Moreimportantly,forFM1-43takenupbyendocytosis,mem-branetraffickingandexocytosiscouldkeepbalanceinaratherlongtimeinvitro(21d).SothequantificationforFM1-43loadingthroughionchannelscouldbemadebythispersistence.AnothersignificantadvantageofFM1-43isitsspecificityforsensorycells.Itpresentsanopportu-nitytovisualizesensorycellsandsensorytransductionundernormalconditionswithminimaldisruptiontothecells.AsIGLEshavebeensupposedtomediatetheiractionviamechanosensitivechannels[9],itwashyp