胞外ATP在男性生殖道中的作用

胞外ATP在男性生殖道中的作用 ActaPhysiologicaSinica,August25,2007,59(4):487-494 http"ffwww.actaps.com.cn TheroleofextracellularATPinthemalereproductivetract ZHOUWen—Liang,,ZUOWu—Lin,RUANYe—Chun,WANGZhe,DUJian— Yang,XIONGYuan,CHANHsiao— Chang DepartmentofPhysiology,SchoolofLifeScience,SunYat— SenUniversity,Guangzhou510275,China;2EpithelialCellBiology ResearchCenter,DepartmentofPhysiology,FacultyofMedicine,TheChineseUniversityofHongKong,HongKong,China Abstract:Inadditiontoitswellestablishedroleasaneurotransmitter,extracellularATPhasbeenconsideredasaparacrine/autocrine factor,eitherreleasedfromspermorepithelialcells,inthemalereproductivetractandshowntoplayaversatileroleinmodulating variousreproductivefunctions.ThisreviewsummarizesthesignalpathwaysthroughwhichATPinducesanionsecretionbythe epitheliaoftheepididymis,aswellasitsepithelium— dependentmodulationofsmoothmusclecontractionofthevasdeferens.Finally, theoverallroleofATPincoordinatingvariousreproductiveeventsinthemalegenitaltractisdiscussed. Keywords:ATP;epidid)mis;vasdeferens;epithelialsecretion;smoothmusclecontraction 胞外ATP在男性生殖道中的作用 周文良?,,左武麟?,阮哗纯?,王站?,杜建阳?,熊原?,陈小章: .中山大学生命科学学院生理学教研室,广州510275;:香港中文大学医学院生理学 系上皮细胞生物学研究中心,香港 摘要:胞外ATP除了能广泛作为神经递质外,还被认为是一种旁分泌或自分泌因 子.ATP从男性生殖道中的精子或上皮 细胞中释放,在调节各种生殖生理功能中起多种作用.本文综述了ATP调节附睾上 皮细胞阴离子分泌的信号通路,阐述了 ATP对依赖上皮细胞的输精管平滑肌收缩的调节机制,讨论了ATP在男性生殖道 中的功能和作用. 关键词:ATP;附睾;输精管;上皮分泌;平滑肌收缩 中图分类号:Q492 1Introduction 1]hemalereproductiveffactcomprises.inad~fiontothe testis,anumberofaccessorysexorgans,includingthe epididymis,vasdeferens,seminalvesicleandprostate, whichalecruciallyimportantformammalianreproduction. Afterleavingthetestis,spermatozoaareneitherableto swimnortofertilizetheegg.Theacquisitionofthese abilities,termedspermmaturation.isachievedbytheir~'an- sitthroughthemaleandfemalereproductivetracts.Inthe epididymis,whichcouldbedividedintothecaput(head), corpus(body)andcauda(tail)regions,spermprogres— sivelyacquirestheirabilitytoswimwitl1forwardmoti— lity?.111eepididymisisthoughttoplayacriticalrolein spermmaturationbycreatinganoptimalfluidmicroenvi— ronmentt*'71.Underbasalconditions.theepididymisreab— sorbsamajorpartofthetesticularfluidandtheabsorptive fluxpredominatesoverthesecretoryfluxts,6,8.?.However. undercertaincircumstances,theepididymiscouldalsobe stimulatedbyphysiologicalregulatorstosecreteionsand otherfactors,importantfordrivingfluidsecretionanden— ablingspermtoobtaintheirmotilityt6,n-141. 1]hevasdeferensisanarrowtractconsistingofbothan epithelialliningandathicklayerofsmoothmusclewitl1 strongcapabilityofcontractionwhichgreatlyfacilitates spemlejaculation.Baseonmorphology,histochemistryand Received2007--07??03Accepted2007??07??09 ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(No.3037054 0,30570229)andtheSouthChina NationalResearchCenterforIntegratedBiosciencesinCollaborationwithZhongshanUniv e~ity. 'Correspondingauthor.Tel:+86-20— 84110060;Fax:+86-20-84110060;E-mail:billzhou200588@yahoo.com.cn 488 electrophysiologicalstudies【l5_.thevasdeferensepithe— liumappearstobesimilartotheepididymalepithelium,and similariontranspoftactivitiesareassumedalthoughno detailedstudieshavebeenreported[2~].Duringejaculation, largequantityofnoradrenalin(NE),withATPasaco-neuro- transmitterTM】.iSreleasedthroughsympatheticnerveend一 gswhichinitiatescoordinatedcontractionofthevasdefe— rensandejectsoutthesperm. 册haslongbeenregardedasanindispensablesource ofenergyforeverysinglecel1.In1953.whenHoltoneta1. foundATPbeingreleasedfromthesynapse【221.itssignifi— canceasaneurotransmitterhasgmnedrecognitionever since.Inadditiontobeingreleasedfromneuronalcells, isalsoknowntobereleasedfromnon—neuronalcells aswel1.includingplatelets[..epithelialcells[26]and basophlilicleukocytes[271.Byusingbioluminescence,we haveshownthatepididymalcellsarecapableofreleasing AexclusivelYintotheapicalcompartmentdrthelumen oftheepididymis(authors'unpublisheddata).Spermalso containhipintracellularlevelof【28.29]andtheconcen— trationofPinthesampleofseminalfluidisconsidered t0beoneofthemostimportantparametersevaluatingthe fecundityofthesperm.Apartfromasthedirectsourceof energyforspermmotilityandfertilization[.rPisalso suggestedtobereleasedfromspermduringtheirtransit throughthemalereproductivetracttoregulatetheirown microenvironmentt33].Therefore.extracellular册mayact asaneurotransmitter,aswellasparacrine/autocrineinthe malereproductivesystem. Inthisreview,wewilldiscusstheroleofextracellular inregulatingthefunctionsoftheepididymisandvas deferens,particularlyepithelialsecretionandsmoothmuscle contraction. 2ATPandepididymalepithelialionsecretion Similartootherepithelia,theabsorptiveandsecretoryac— tivitiesofepididymalepitheliumareregadatedormodulated bymanyfactors.suchasneurotransmitters['3?34-3.hor— monesandparacrine/autocrineagents[39】. Itwasproposedalmosttwodecadesagothatepididy— malspermatozoacouldcontroltheirownfluidenviron— mentbyreleasingATPintothelumenofepididymistoregu- latethesecretoryactivityoftheepididymis[451.Thiswas basedonthefirstobservationoftheeffectofextracellular Ponepididymalepithelium[46].AddingextracellularP totheapicalbutnotbasolateralsideoftheprimaryculture ofratepididymialmonolayercouldstimulateashort— circuitcurrentresponse.consistingofa缸ansientspike ActaPhysiologicaSinica,August25,2007,59(4):487-494 followedbyasecondsustainedresponse.Thefirstand secondpeaksoftheA一inducedcurrentresponsewere dose—dependentandbothreachedamaximallevelat10 iMno1]L[46.47】.Thetwopeakswerediminishedbyremoval OfCrfrombathingsolution,indicatingthattheATP— inducedcurrentresponsewasmainlyduetoCI.secretion[45】. ThebiphasiccurrentresponsetoAcouldbeattributed totheinvolvementofdifferentpyrinergicteeeDtorslead— ingtodifferentregulatorypathwaysandthusdifferentcur- rentkinetics.InhibitorofP1receptors,8-phenyltheophylline wasfoundtohavenoeffectsonthe.stimulatedcurrent. 1[1leeffectsofotheradenosinederivatives.purineandPY— rimidinenucleotideswerealsodetectedfortheirpotency instimulatingtheshort—circuitcurrent,whichhavethe followingsequenceoforder:ATP>ADP>>AMP- adenosine.andA1rP>D以P>GTP>CTP.Thiswascon— sistentwiththenotionthatP,Y,butnotP1-purinoceptors wereinvolvedinmediatingtheATPresponset46'47】.Therefore, thebiphasicnatureofthe.inducedcurrentresponse wasnotlikelyduetotheinvolvementofdifferenttypesof purinoceptors.Pretreatmentofthecultureswithpiroxicam didnotaffecttheresponsetoATP,butabolishedtheshort- circuitcurrentresponsetobradykinin,whichindicatesthat theshort-circuitcurientresponsetoAwasnotmediated byanincreaseinthesynthesisofprOstaglandins【461. Disulfonicacidstilbene(DIDS),whichhadbeenfoundto haveaninhibitoryeffectonCa2~一activatedCl.channe1. inhibitedthefirst,butnotthesecond,peakoftheATP inducedcurrentwhilediphenylamine—dicarboxylicacid (DPC).whichisanon—specifcC1.channelblocker.re— ducedb0tIl【471.Itappears.therefore.thatthetwophases oftheATP—inducedcurrentresponsearelikelytoinvolve differenttypesofCl_channels.whichsuggeststhatdiffe— rentsecondmessengersmaybeactivatedbvmRadio— immunoassay(RIA)measurementsshowedthatintracel— lularcAMproductionwasincreasedbyextracellularATP, butnotadenosine[47].AlsO.itwasobservedthatATPeli— citedariseinintracellularCa2.intheepididymalepithelium dependentmannert4~].1[1leseresultssug. inaconcentration— gestbothcAMandCa2~pathwaysaleinvolvedinmedi— atingtheeffectofrPinstimulatingCl.secretion.Further short-circuitcurrentexperimentsshowedthatB effectivelyinhibitedbothpeaksoftheP.inducedcurrent, butitdidnotaffecttheforskolin—inducedcurrentresponse. AlsotheK.inducedcproductionwasprevented bypretreatmentwitIlBAPTA/AM【47】.Theseresultssug— gestedthattheP.inducedcAMPactivationwasse. condarytoanincreaseinintraceUularCa2+concentration. Inordertoconfirmtheinvolvementofdifferenttyl~sof ZHOUWen—Liangetal:RoleofExtracellularATPintheMaleReproductiveTract C1.channels.namelytheCa2一andcAMP—dependentCl— conductances,inmediatingthestimulatoryeffectofATP, thewhole—cellpatch-clamptechniquewasconducted.Two currentkineticswereobserveduponKstimulationde— pendingonthetimecourse.Oneexhibiteddelayedinacti— vationandactivationkineticsathyperpolarizinganddepo— larizingvoltages,respectively,whichweresimilartothat oftheCa2+一activatedCl—channe1.Atalatertimepointafter ATPstimulation.anotherwhole—cellcurrentprofilewas observedexhibitingtime..andvoltage..independent characteristic.similartothecAMP-activatedC1.conduc— tallce[49—5".nowknowntobemediatedbythecysticfibro— sistransmembraneconductanceregulatorfCFIR).DIDS couldonlYdiminishthecurrentexhibitingvoltage..depen.. dentcharacteristicwhileDPCinhibitedbothtypesofcur- rentscompletely/49].1]heseresultsareconsistentwiththat observedbytheshort-circuitcurrentexperiments[491.iIldi— caringtheinvolvementofdifferentC1.channels.Intere— stingly,pretreatmentOfPKI,inhibitorforproteinkinaseA, blockedtheATP—inducedtime—andvoltage—independent currentactivation,butnotthedelayedactivatingandinac— tivatingcurrentactivationwhichwasDIDS—sensitive; however.aCa2chelator,EG1'A.inthepipettesolution couldsuppressbothtyl~softheAI'P.activatedcurrents[491. Furtherexperimentsblockingthe一coupledsignaling pathwayupstreamofCamobilization(i.e.,byheparin, aninhibitorofIP)orinhibitingthekeyCa2一dependent enzymethatmightberesponsibleforactivatingadenylate ATP cyclaseandthuscAMP(i…ebytrifluoperazine,aninhibitor ofcalmodulin)resultedincompleteinhibitionofboth— activatedCa2+一andcAMP-dependentCl—currents[49].These resultssuggestthattheATP—inducedactivationofcAMP- dependentpathwayissecondarytotheCa2+_dependent pathway,whichisconsistentwiththeresultsobtainedfrom theshort-circuitcurrentmeasurementorR工A(seeabove). Takentogether,asignaltransductionmodelhasbeen proposedfor.inducedCa2.andcAMP-dependentac. tivationofCrsecretion.asshowninFig.1.Itsuggests thatbOtllCa2+一andcAMP—dependentC1.channelsarein— volvedinmediatingtheATP—stimulatedCrsecretion.in— volvingP2Y2receptors,IP3一dependentCarelease,acti— vationofcalmodulinwhichthenactivatestheCa2+-depen— dentCrchannelontheonehandandinteractswiththe adenylatecyclaseontheotherhand,leadingtoactivation OfcAM[f).dependentCrchannelandthusCl-secretion. ApartfromitseffectontheC1.channels'activationob— servedinratepididymalcells.ATPhasalsobeenshownto activateal(十conductanceinhumanepididymalcellsI52].In theepididymalfluid,theI(+contentalongthelengthof epididymisincreasesprogressivelyandreachesitsmaxi— muminthecaudaregionf一.whichisthoughttobedue toKsecretion【531.Ithasbeensuggestedthatsomerectifying cationconductanceisthereasonforthehighI(+secre— tionf0,.A1so.microperfusiontechniqueinvivowascar- riedouttostudytheKsecretionandithasbeendemon. stratedthattheepididymisismainlyresponsibleforthe Fig.1.ModelforextracellularATP-inducedsignaltransducfionpathwaysinratepididymalc eHs.ATPactivatesP2Y2receptortoproduceIP3, whichincreasesintracellularCa2~content.Ca2thenbindstocalmodulinandactivatesCa2~- dependentC1'channe1.Activatedcalmodulinmay interactwithadenylatecyclasetoproducemorecANIP.whichactivatescAMP-dependentC 1'channelthroughthePKApathway.The activationofbothCa2~_andcAMP-dependentC1'channelsisresponsibleformediatingthes timulatoryeffectofATPonC1'secretion. highK+contentintheluminalfluid【6】.Anoutwardlyrecti— fyingcationconductance,whichmayberesponsiblefor thehighKsecretion,wasobservedintheprimarycul— turedhumanepididymalcells.TIlisoutwardlyrectifying cationconductancewasnotaffectedbyremovalofthe intracellularandextraceUularC1.andNa+.butwasmainly blockedbychangingtheintracellularbutnotextracellular K+concentration.indicatingKselectivityofthisconduc— tance[52]. TIlisconductancecouldalsobeinhibitedbyca— tionchannelblockers,bariumandtetraethylammonium,and mostinterestingly,thiscationconductancecouldbeacti— vatedbyextmcenularArPThissuggeststhat册released fromtheepitheliumorthespermatozoamakingtheirway throughtheepididymismayactasaregulatorofthefluid environmentsurroundingthespermbypromotingKse- cretion【j.althoughthephysiologicalsignificanceofsuch ahighKcontentonspermfunctionremainstobe elucidated.Ontheotherhand.activationofaKconduc. tallcemayfacilitateanionsecretion[6o],ratherthanK+ secretion.iftheKchannelislocalizedtothebasolateral membranetoprovideadrivingforceforanionexitatthe apicalmembrane[61(alsoseereviewbyBleichandShanin thisIssue).Indeed.addingKchannelblockerstothepri. maryculturesofratepididymalepithelialcellscauseda concentration-dependentinhibitionoftheadrenaline-induced anionsecretion[39].indicatingtheinvolvementofKchan. nelinanionsecretionacrosstheepididymalepithelium. Furtherstudiesarerequiredtodeterminethelocalization oftheAIP.activatedKchannelinordertoascertainits functionalrole,inKsecretionoranionsecretion. Takentogether,theimportantroleOfArPinregulating thefluidenvironmentoftheepididymisisevidenced. Similarly,A:rPcanalsostimulateanionsecretionacross theprimarycultureofporcinevasdeferensepithelium[2~]; however,thedetailsregardingthereceptorsandsignaling pathwaysinvolvedrequirefurtherstudies. TIlemajorfunctionofthevasdeferensistotransportand excretesperm.Theinnervationofthevasdeferenshas beenshowntobemostlysympatheticwithNEasthema. {orneurotransmitter[21】.However;thereisalsoabundant evidenceindicatingthatA:rPalsoplaysaroleinsympa- theticneuroeffectormechanismsasacotransmitterwith NE【1,62-651. Earlyelectricalfieldstimulation(EFS)ofthesympathetic nervesinthevasdeferensresultedinacontractionwith twodistinctcomponents,includingarapidpeal(followed ActaPhysiologicaSinica,August25,2007,59(4):487-494 byamaintainedcontractionforthedurationofthe stimulation.TIlephenomenonofthebiphasiccontraction thussuggestedpossibleinvolvementofdifferentneuro- transmiRers.Furtherinvestigationsrevealedthatboth andNEwerereleasedfromnervesinnervatingtheratvas deferensduringfldstimulation[66].Tetrodotoxin(TTX) andguanethidineabolishedbothATPandNEoverflow whereaspretreatmentwithreserpineabolishedtherelease OfNEbutnotAZp[67】.indicatingthatthetwoneurotrans. mitterswerereleasedseparately.Itisnowcleartl1atthe firstphaseoftheresponseismediatedmainlybyact- ingonpostjunctionalP2Xreceptors,whereasthesecond phaseismediatedmainlybyNEactingon%-adIoce)rs, whichisincontrarytotheATP.inducedbiphasicanion secretoryresponseintheepididymalepithelium,involving singlereceptorbutdifferentsecondmessengers. IthasbeensuggestedthatAmayalsofunctionasa neuromodulator,whichcausesareductionofneumtransmit- terrelease[64,69].Thestudywithantagonistsofreceptors foradenosinesuggestedthattheeffectofAwasme. diatedbyitshydrolysisofadenosinethusactivatingP1re. ceptorstoinhibittheneurotransmitterrelease[70].Adeno. sinehaslongbeenknowntoreduceneurotransmitterre. 1easeinthevasdeferensandothertissuesbutthespecific typesofreceptorinvolvedhavenotbeenfLll1velucidatedt6~1. IthasbeenestablishedthatA:rPcanalsoactasanago. mstoftheP2Xreceptorsinthevasdeferens.Theextrace1. 1ularregulationofsynaptictransmissionwouldevokethe contractionofsmoothmuscle.Recentstudyrevealedthat P,XreceptorsaremorepermeabletoCa2comparedto otherionchanne1.AstheagonistofP,Xreceptor.A wouldchangethetransformationofthereceptoronthe membraneandfacilitatetheinfluxofcations.thedepolari. zationofthemembranepotential,increaseintheperme. abilityofmembraneandthusevokethecontractile responses.TheA-inducedsmoothmusclecontraction ismediatedviaP2Xreceptorslocatedonthevasdeferens smoothmuscle,whichareligand.gatednon.selectiveca. tionchannelsandwhenactivatedproduceandsummate theexcitatory{unctionpotentialscausingsmoothmuscle contractiont7~】. Meanwhile,气hasbeenobservedtohaverelaxanteffect. Inthecardiovascularsystem,ATPi