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Opioidergic Modulation of Luteinizing and Growth Hormone Release From Opioidergicmodulationofluteinizingandgrowth hormonereleasefromporcineadenohypophysesinvitro VondemFachbereichBiologiederUniversitätHannoverzur ErlangungdesGradeseinesDoktorsderNaturwissenschaften Dr.rer.nat. GenehmigteDissertation von EnowmpeyEnowtambong (B.Sc.,M.Sc.) geborenam26.03.68inFumbe-Mamfe/KAMERUN 2002 Referentin:FrauProf.Dr.Dr.N.Parvizi Korreferentin:FrauProf.Dr.C.Aurich Korreferent:Prof.Dr.S.Steinlechner TagderPromotion:14.06.2002 ThisresearchworkwascarriedoutattheFederalResearchCentrefor Agriculture,FAL,InstituteforAnimalScienceandAnimalBehaviour, Mariensee,31535Neustadt,RepublicofGermany. ABSTRACT EndogenousopioidsareknowntomodifyLHandGHsecretionthroughthehypothalamo- pituitaryaxis.ThisstudyaimedatverifyingthedirectactionofopioidsonLHandGH secretionatthepituitarylevelusingpituitaries(adenohypophyses)ofpigsinaperifusionin vitrosystem.FurthermoretodeterminewhetheropioidergicmodulatedreleaseofLHandGH isalteredbyLHRHorGHRHstimulationatthepituitaryinpigsandiftheseeffectsaresex-, andage-dependent. ß-endorphin(5*10-9M)significantlyincreasedthereleaseofLHinfetuses(425%inmales and275%infemales)piglets(325%inmalesand175%infemales)adults(245%inmales and160%infemaleswithhigh(P>3.5ng/ml)progesteroneconcentration)ascomparedto salinecontrols.Naloxone(10-6M)anddalargin(10-6M)showednoeffects.Surprisingly naloxonedidnotantagonisetheß-endorphin-inducedLHreleasewhenconcomitantly administeredtopituitariesofadultfemalesandmales.Pretreatmentwithß-endorphin attenuatedLHRH-stimulatedLHreleaseinfetalandadultfemalesbutnotmales,leaving pigletsunaffected.NaloxoneanddalarginimpairedLHRH-inducedLHincrementinboth adultsandfetuses.Againnaloxoneanddalarginpreadministrationhadnoinfluenceon LHRH-inducedLHreleaseinpiglets. Theopiateagonists,ß-endorphinanddalargincausedasignificantincreaseinGHsecretion inpituitariesofadultmalesbutnotinfemalesthusindicatingasex-differentialeffect. Whereasnaloxoneshowednoeffectinbothsexes,concomitantadministrationofß- endorphinandnaloxoneshowedantagonisminadultmalesbutnotinfemales.Inpiglets, naloxoneelicitedanincreaseinGHsecretioninfemaleswhereasinmales,anon-significant decreasewasobserved.ß-endorphinanddalarginshowednoeffectsinpituitariesofboth femaleandmaleofpiglets.Ontheotherhand,ß-endorphinincreasedGHsecretion significantlyinpituitariesoffetalmalesbutnotinfemales.Dalarginandnaloxoneshowedno effectsinbothfemaleandmalefetusesunlikeinadultsandpiglets. Pretreatmentwithß-endorphintendedtopotentiateGHRH-stimulatedGHreleasefrom pituitariesofadultmales.Interestingly,posttreatmentwithLHRHdiminishedGHdischarge inadultfemalespretreatedwithß-endorphinandnaloxoneleavingmalesunaffected. ThesefindingsindicatethateffectsofopioidsonspontaneousLHandGHsecretionare evidentinthepigpituitaryandtheseeffectsaresex-,age-andsteriod-dependent.Opioids couldplayamajorroleinmodifyingLHRH-inducedLHreleasebutexertminoreffectsin modulatingGHRH-inducedGHdischargefromthepigpituitary. Keywords:OPIOIDS,HORMONES,PIG ABSTRACT EndogeneOpioidemodulierendieSekretionvonLHundGHhauptsächlichüberdieHypothalamus- Hypophysenachse.ImRahmendieserArbeitsolltederEinflußvonOpioidenaufdieLHundGH SekretionaufderhypophysärenEbeneineinemSuperfusionssystemuntersuchtwerden.Weiterhin wurdeuntersucht,obOpioidedieLHRHinduzierteLH-Ausschüttungbzw.GHSekretion beeinflussenundobdieEffekteAlters-,undGeschlechtsabhängigsind. AusdenErgebnissengehthervor,dassß-Endorphin(5*10-9)einensignifikantenAnstiegder hypophysärenLHSekretioninFöten(425%beimännlichenvs275%beiweiblichen),beiFerkeln (325%beimännlichenvs175%beiweiblichen)undbeiAdulten(245%beimännlichenvs160%bei weiblichen,miteinerhöherenProgesteronKonzentration=P>3.5ng/ml)verursachte.Naloxon(10-6M) undDalargin(10-6M)dagegenwiesenkeineEffekteauf.DieVorbehandlungmitß-Endorphinführte zuBlockadenderLHRH-stimulierendeLH-Ausschüttungsowohlbeifötalenalsauchbeiadulten weiblichenabernichtbeimännlichenHypophysen,währenddieReaktionderFerkelunverändert blieb.AnderseitshabenNaloxonundDalargindieLHRHinduzierteLH-Ausschüttungbeiadulten undfötalenHypophysennurleichtverdrängt.DieVorbehandlungmitNaloxonundDalarginzeigte dagegenkeinenEffektbeiFerkeln. DieOpioid-Agonisten,ß-EndorphinundDalarginverursachteneinensignifikantenAnstieg derGHSekretionbeiEberabernichtbeiSauen.DiesdeuteteinengeschlechtsdifferenziertenEffekt an,wogegenNaloxonkeinenEffektbeibeidenGeschlechtenaufwies.Naloxonzeigteeinen Antagonismusmitß-EndorphinbeimännlichenabernichtbeiweiblichenadultenHypophysen.Bei denFerkelnführteNaloxonzueinemAnstiegderGHSekretionbeiweiblichen,währendbei männlichennureinenicht-signifikanteVerringerungderGHSekretionbeobachtetwurde.ß- EndorphinundDalarginwiesenkeineEffektebeimännlichenundweiblichenFerkelnauf.ß- EndorphinführtezueinemsignifikantenGHAnstiegbeimännlichenfötalenHypophysen,nicht jedochbeiweiblichen.DalarginundNaloxondagegenzeigtenkeinenEffektaufdieGHSekretion beiweiblichenundmännlichenfötalenHypophysen.DieVorbehandlungmitß-Endorphinhattekeine signifikanteWirkungaufdieGHRH-induzierteGHAusschüttung.Lediglichwareinerelativstarke abernichtsignifikantePotenzierungderGHRH-induzierteGHSekretionbeiEbernzubeobachten. DieBehandlungmitLHRHführtezueinerVerringerungderGHAusschüttungbeiweiblichen Hypophysen,abernichtbeimännlichen,diezuvormitß-EndorphinundNaloxonbehandeltwurden. AusdiesenErgebnissengehthervor,daßOpioidediespontaneLHundGHSekretionaufder hypophysärenEbenemodulieren.DieseEffektesindvonGeschlecht,AlterundgonadalenSteriode abhängig.OpioidekönneneinebedeutendeRollebeiderModulationderLHRH-induziertenLH Ausschüttungspielen,abersiezeigeneinengeringenEffektbeiderModulationderGHRH- induziertenGHSekretionderSchweinhypophysen. SCHLAGWORTE:OPIOIDE,HORMONE,SCHWEIN TABLEOFCONTENT PAGES 1.0 INTRODUCTION………………………………………………………...1 1.1 LiteratureReview...................................................................................…....2 1.2 ClassificationandCharacterizationofEndogenousOpioidsandtheir receptors.................................................................................................…....2-14 1.3ThemechanismofOpioidLigand-Receptorinteractioninmodification ofcellfunction..............................................................................…………14 1.4.0 EndogenousopioidsandLuteinizingHormoneSecretion................…..15-18 1.4.1 TheeffectsofendogenousopioidsonLuteinizingHormonesecretion fromfetaltopubertalstagesinthepig..................................................…....19-20 1.4.2 TheeffectsofendogenousopioidsonLuteinizingHormonesecretion intheadultpig.....................................................................................…….20-23 1.4.3 InvitroeffectsofopioidsonLuteinizingHormonesecretion..............…...23-25 1.5.0 EndogenousOpioidsandGrowthHormoneSecretion...................…..25-26 1.5.1 TheeffectsofendogenousopioidsonGrowthHormonesecretion fromfetaltopubertalstages...............................................................……..26-29 1.5.2 TheeffectsofendogenousopioidsonGrowthHormonesecretion inadults.............................................................................................……...29-33 1.5.3 InteractionofopioidsandGHRHinthecontrolofGrowthHormone secretion............................................................................................……..33-34 2.0AIMSANDOBJECTIVES...................................................…………….35 3.0 MATERIALSANDMETHODS...........................................………..36 3.1 ExperimentalAnimals......................................................................………36 3.2 Removalofanteriorpituitary.............................................................…….36-37 3.3 Perifusion...........................................................................................……..37-38 3.4 Reagents..............................................................................................…….39 3.5 ExperimentalDesign..........................................................................…….39-41 3.6 HormoneAnalysis..............................................................................…….41 3.6.1 LuteinizingHormoneAnalysis............................................................……41-42 3.6.2 GrowthHormoneAnalysis...................................................................…...42 3.6.3 ProgesteroneAnalysis.......................................................................……42 3.7 StatisticalAnalysis...............................................................................…...43 4.0 RESULTS.............................................................................…...……….44 4.1.0 OpioidergiceffectsonLHrelease......................................................….45 4.1.1 OpioidergiceffectsonLHreleaseinfetuses........................................….45 4.1.2OpioidergiceffectsonLHreleaseinpiglets.......................................….45-48 4.1.3 OpioidergiceffectsonLHreleaseinadults........................................…..49-53 4.2.0Opioid-LHRHinteractionandLHrelease…....................…………..54 4.2.1Fetuses..................................................................................................….54 4.2.2Piglets..................................................................................................…..54-57 4.2.3Adultfemales......................................................................................…..58-59 4.3.0 OpioidergiceffectsonGHsecretion...............................................…...59 4.3.1OpioidergiceffectsonGHsecretioninfetuses.................................……59-61
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